Research Article

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity

Department of Chemistry and Biochemistry, University of Oregon, United States
Institute of Molecular Biology, University of Oregon, United States
Department of Chemistry, University of California, Berkeley, United States
California Institute for Quantitative Biosciences, United States
Department of Molecular and Cell Biology, United States

Aug 17, 2022

https://doi.org/10.7554/eLife.73747

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Version of Record published: September 13, 2022 (This version)
Accepted Manuscript published: August 17, 2022 (Go to version)
Accepted: August 16, 2022
Preprint posted: September 22, 2021 (Go to version)
Received: September 9, 2021

1. Of interest
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Research Article May 25, 2023
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Abstract

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P₂ lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P₂ lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer–dimer equilibrium. PIP5K monomers can associate with PI(4,5)P₂-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P₂ binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P₂ and membrane-bound kinase.

Editor's evaluation

This study presents highly interesting and detailed biochemical analyses into the mutual relationship between PI(4,5)P₂ lipids and their kinase PIP5K, which engage in an exciting pattern-forming reaction on membranes. Using direct single-molecule imaging approaches, the authors find cooperative recruitment of PIP5K to the membrane, dimerization-enhanced catalytic efficiency and indications of allosteric regulation. This article is of interest to a wide range of readers who study the biology of lipid-modifying enzymes, especially as it relates to interfacial reaction kinetics in biological membranes.

https://doi.org/10.7554/eLife.73747.sa0

Introduction

Phosphatidylinositol phosphate (PIP) lipids are an important class of second messengers that regulate the localization and activity of proteins on every intracellular membrane in eukaryotic cells (Di Paolo and De Camilli, 2006; Balla, 2013). Synthesis of PIP lipids is regulated by different classes of lipid kinases and phosphatases that drive the interconversion between PIP lipid species through the phosphorylation and dephosphorylation of inositol head groups. Of particular importance to a vast array of signaling pathways are phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] lipids, which comprise a minor phospholipid component of the total cellular membrane composition (i.e., ~2 %) (Wenk et al., 2003; Nasuhoglu et al., 2002). PI(4,5)P₂ serves many important functions in biological processes, including receptor activation, ion channel function (Hansen, 2015), endocytosis (Zoncu et al., 2007; Jost et al., 1998), and actin network assembly at the plasma membrane (Janmey et al., 2018). Understanding the mechanisms that control PI(4,5)P₂ lipid synthesis is critical for deciphering how cells regulate receptor signaling and PIP lipid homeostasis at the plasma membrane.

Two classes of lipid kinases catalyze the production of PI(4,5)P₂ lipids: phosphatidylinositol 4-phosphate 5-kinase (PIP5K, type I PIPK) and phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK) (Burke, 2018). These two families of lipid kinases differ in substrate specificity; PIP5K phosphorylates phosphatidylinositol 4-phosphate – PI(4)P, while PIP4K phosphorylates phosphatidylinositol 5-phosphate – PI(5)P (Loijens and Anderson, 1996; Rameh et al., 1997; Muftuoglu et al., 2016). Due to the higher abundance of PI(4)P in the plasma membrane, relative to PI(5)P, the majority of PI(4,5)P₂ lipids are generated from reactions catalyzed by PIP5K (Doughman et al., 2003; Balla, 2013). Functionally conserved across eukaryotes, yeast express a single PIP5K enzyme denoted Mss4 (multicopy suppressor of Stt4) (Yoshida et al., 1994; Homma et al., 1998; Desrivières et al., 1998). In humans, three paralogs – PIP5KA, PIP5KB, and PIP5KC – regulate the production of most PI(4,5)P₂ lipids found at the plasma membrane.

Several mechanisms regulate membrane docking of PIP5K, including substrate recognition (Muftuoglu et al., 2016; Kunz et al., 2000), electrostatic interactions with anionic lipids at the cell plasma membrane (Fairn et al., 2009), and membrane binding of an amphipathic helix (Liu et al., 2016). Single-molecule characterization of human PIP5KB has revealed a role for PI(4,5)P₂ lipids in controlling cooperative membrane association and positive feedback during PI(4)P lipid phosphorylation reaction (Hansen et al., 2019). Structural and biochemical studies indicate that, like PIP4K (Rao et al., 1998; Burden et al., 1999), PIP5K can homodimerize in solution (Hu et al., 2015). In the case of zebrafish PIP5KA (zPIP5KA), dimerization has been shown to be required for lipid kinase activity (Hu et al., 2015). It remains unclear whether dimerization regulates membrane docking, ATP binding, or catalysis of PIP5K. Overall, the sequence of molecular interactions that control PIP5K membrane localization and activation has not been elucidated.

Using total internal reflection fluorescence (TIRF) microscopy to measure the kinetics of PIP lipid phosphorylation on supported lipid bilayers (SLBs) in vitro, we previously reported that human PIP5KB catalyzes the phosphorylation of PI(4)P with a positive feedback loop based on association with its reaction product, PI(4,5)P₂ (Hansen et al., 2019). Based on the crystal structure of zebrafish PIP5KA (Hu et al., 2015; Muftuoglu et al., 2016) and previous biochemical data, we have worked under the assumption that members of the PIP5K protein family function as obligate dimers. However, through comparative single-molecule TIRF (smTIRF) microscope measurements of PIP4K and PIP5K membrane-binding dynamics in vitro we discovered that members of the PIP5K protein family exist in a monomer–dimer equilibrium in solution. At low molecular densities, PIP5K protein family members can associate with PI(4,5)P₂ membranes as a monomer and catalyze the phosphorylation of PI(4)P. Under these conditions, the mechanism of PIP5K-positive feedback is controlled by cooperative binding to the reaction product, PI(4,5)P₂. Increasing the surface density of membrane-bound PIP5K promotes dimerization, which further increases the dwell time and enhances the catalytic efficiency of the kinase ~20-fold. Consistent with a mechanism of allosteric regulation, dimerization can increase PIP5K catalytic efficiency independently of enhancing membrane avidity. We find that the increase in kinase activity afforded by membrane-mediated dimerization – more specifically the strong positive feedback it creates – dramatically enhances the PIP5K’s ability to form bistable PIP compositional patterns in the presence of an opposing PIP lipid phosphatase on SLBs. The membrane-mediated dimerization also amplifies stochastic fluctuations in kinase reaction velocity. In the context of spatial confinement, these magnified fluctuations facilitate mechanisms such as the recently reported stochastic geometry sensing, in which bistability and even the deterministic outcome of a competitive reaction may depend on system size (Hansen et al., 2019; Lee et al., 2021). Together, our results highlight a mechanism by which PI(4,5)P₂ binding and membrane-mediated dimerization create a broad dynamic range of PIP5K activities that cells can potentially leverage to tune the concentration and spatial distribution of PI(4,5)P₂ lipids on cellular membranes.

Results

PIP4K and PIP5K bind to PI(4,5)P₂ membranes with distinct oligomerization states

The PIP4K and PIP5K families of lipid kinases both reportedly form homodimeric complexes, but with structurally distinct dimer interfaces (Figure 1A; Hu et al., 2015; Rao et al., 1998; Burden et al., 1999). Consistent with size-exclusion chromatography and multi-angle light scattering (SEC/MALS) data published by Hu et al., 2015, we found that wild-type and the dimerization-deficient zebrafish PIP5KA (zPIP5KA) mutant, D84R, showed distinct SEC elution profiles that reflect the dimeric and monomeric states of each kinase (Figure 1—figure supplement 1). However, comparing the SEC elution profiles of zPIP5KA and PIP4KB – both predicted to have dimeric molecular weights of 90 kDa – indicated that zPIP5KA eluted slowly compared to PIP4KB and protein molecular weight standards (Figure 1—figure supplement 1). Differences in the elution profile of zPIP5KA and PIP4KB could be the result of distinct subunit orientations or the oligomerization state. This observation led us to further investigate whether the PIP5K family of proteins generally exists in a monomer–dimer equilibrium, rather than being obligate dimers like PIP4K. Unfortunately, our attempts to measure the strength of PIP5K dimerization in solution using fluorescence polarization anisotropy and analytical ultracentrifugation were unsuccessful. For this reason, we established a single-molecule imaging approach to directly visualize and quantify the propensity of PIP4K and PIP5K proteins in solution to associate with PI(4,5)P₂-containing membranes as either a monomer or dimer. Deciphering the relationship between PIP5K membrane binding, oligomerization, and catalysis, we describe here the molecular basis of positive feedback during PIP5K-dependent generation of PI(4,5)P₂ lipids.

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PIP4K and PIP5K can bind to PI(4,5)P₂ membranes with distinct oligomeric states.

(A) Cartoon showing the kinase domain orientation and proposed oligomerization states of PIP4K and PIP5K homodimers. PIP5K potentially exists in a monomer–dimer equilibrium, while PIP4K is a constitutive dimer. (B) Single-molecule total internal reflection fluorescence (TIRF) microscopy images of supported lipid bilayers (SLBs) incubated with either 1 pM mNG-PIP4KB and 5 pM mNG-PIP5KB. (C) Intensity line scans through single mNG-PIP4KB and mNG-PIP5KB molecules bound to membranes in (B). (**D, E**) Single-molecule photobleaching dynamics of mNG-PIP4KB (red line) and mNG-PIP5KB (red line) compared to background fluorescence (black line). (**F, G**) Dynamic of change in molecular brightness frequency distribution of mNG-PIP4KB and mNG-PIP5KB during photobleaching. Each histogram represents the population distribution measured during a 1 s window over the course of a 10 s photobleaching experiment. N = 1300–1500 mNG-PIP4KB particles per second (F and Figure 1—video 1), N = 200–300 mNG-PIP5KB particles per second (G and Figure 1—video 1). (H) Probability of observing mNG-PIP4KB and mNG-PIP5KB dimers at different times during the photobleaching experiment shown in (**F, G**). The threshold particle intensity was ≥8200 (A.U.) to be considered a dimer from data in (**F, G**). Note that the fraction of mNG-PIP4KB dimers does not approach 0% because there are new membrane-binding events during the image acquisition. (I) Molecular brightness distributions measured in the presence of mNG-tagged mouse PIP5KA (5KA), human PIP5KB (5KB), human PIP5KC (5KC), zebrafish PIP5KA (z5KA), yeast Mss4 (yMss4), and human PIP4KB (4KB) (N = 3911–23304 particles per distribution). (J) Step-size distribution plots generated from single-molecule tracking in the presence of mNG-tagged 5KA (D = 0.214 µm²/s), 5KB (D = 0.191 µm²/s), 5KC (D = 0.165 µm²/s), z5KA (D = 0.234 µm²/s), yMss4 (D = 0.199 µm²/s), and 4KB (D = 0.051 µm²/s) (N > 10,000 total steps [or displacements in µm] per distribution derived from 2 to 3 technical replicates). (**B–J**) Membrane composition: 96% DOPC, 4% PI(4,5)P₂. Note that the percentage of mature and fluorescent mNG fusions is ~80% for each labeled kinase.

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To determine whether PIP4K and PIP5K proteins in solution can be recruited to membranes with distinct oligomerization states, we established a single-molecule cell lysate assay (Lee et al., 2017) to compare the membrane-binding properties of fluorescently labeled human PIP4KB and PIP5KB (referred to as 4KB and 5KB in figures) on SLBs using smTIRF microscopy. For these experiments, genes encoding mNeonGreen (mNG) fused to either PIP4KB or PIP5KB were transiently expressed in human embryonic kidney (HEK) 293 cells. Cells were lysed by sonication and centrifuged to remove membranes and debris. We quantified the concentration of mNG-PIP4KB and mNG-PIP5KB in clarified cell lysate using a purified mNG protein standard (Figure 1—figure supplement 2). Samples containing mNG-labeled kinase were then diluted ~10,000-fold in imaging buffer to a concentration of 10 pM and incubated on supported membranes containing 4% PI(4,5)P₂ lipids. Under these conditions, the resulting surface density of membrane-bound mNG-PIP4KB and mNG-PIP5KB was ~0.03 molecules/µm² (or ~100 molecules per field of view) (Figure 1B).

To quantify the differences in the oligomerization states of membrane-bound mNG-PIP4KB and mNG-PIP5KB, we compared the molecular brightness and diffusion coefficients of single particles by smTIRF microscopy. Intensity line scans through mNG foci revealed that the majority of mNG-PIP4KB molecules were two times brighter compared to membrane-bound mNG-PIP5KB (Figure 1C). Because mNG-PIP4KB is a obligate dimer (Rao et al., 1998; Burden et al., 1999), the molecular brightness distribution of this lipid kinase set the upper limit for the percentage of detectable dimers in our assay. This was based on the fraction of mNG molecules that formed mature chromophores during expression in HEK293 cells, which was ~80% for mNG-PIP4KB. To determine the oligomerization state of membrane-bound mNG-PIP4KB and mNG-PIP5KB, we performed single-molecule photobleaching experiments. In the case of mNG-PIP4KB, the vast majority of membrane-bound particles photobleached in two steps (Figure 1D). By contrast, single-membrane-bound mNG-PIP5KB particles photobleached in a single step (Figure 1E).

To measure changes in the molecular brightness distribution, we continuously monitored the fluorescence of either membrane-bound mNG-PIP4KB or mNG-PIP5KB under conditions that promoted stepwise photobleaching. Over the course of a 10 s photobleaching experiment, the molecular brightness distribution for mNG-PIP4KB shifted from bimodal to a single peak (Figure 1F, Figure 1—video 1). During this time, the fraction of mNG-PIP4KB dimers with two visible mNG fluorophores gradually decreased from ~80% to ~20% of the population (Figure 1H). By contrast, the molecular brightness distribution of mNG-PIP5KB remained unimodal throughout the 10 s photobleaching experiment, consistent with mNG-PIP5KB associating with PI(4,5)P₂-containing membranes predominantly as a monomer at a low protein surface density (Figure 1G, Figure 1—video 2). We also observed similar differences in molecular brightness comparing recombinantly expressed and purified PIP4KB and PIP5KB that were chemically labeled with Alexa488 in vitro using Sortase-mediated peptide ligation (Figure 1—figure supplement 3). However, the lower labeling efficiency achieved using Sortase-mediated peptide ligation led us to measure the molecular brightness distribution of PIP5K homologs and paralogs using exclusively mNG fusion proteins in this study. Comparing the molecular brightness distributions of mNG-PIP5KA (mouse), mNG-PIP5KB (human), mNG-PIP5KC (human), mNG-zPIP5KA (zebrafish), and mNG-yMss4 (yeast multicopy suppressor of Stt4 or yeast phosphatidylinositol 4-phosphate 5-kinase), we also observed only single peaks corresponding to monomeric kinases labeled with a single fluorescent mNG (Figure 1I). Photobleaching analysis of membrane-bound mNG-PIP5KA and mNG-zPIP5KA produced similar single-step photobleaching results to those observed for mNG-PIP5KB (Figure 1—figure supplement 4).

Consistent with mNG-PIP5K and mNG-PIP4K binding to membranes as either monomers and dimers, respectively, the two lipid kinases also exhibited strikingly different diffusivity when membrane bound. At low surface densities (~0.01 molecules/µm²), the mobility of mNG-PIP4KB (0.04 µm²/s) was much slower compared to mNG-PIP5K homologs and paralogs (0.15–0.18 µm²/s) (Figure 1J). These diffusion coefficients were very similar to that measured for PIP5KB labeled with an Alexa647 chemical dye (Figure 2C), indicating that membrane binding of the mNG kinases in dilute cell lysate is indistinguishable from recombinantly purified and fluorescently labeled enzymes.

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Protein density-dependent changes in PIP5K membrane binding.

(A) Supported lipid bilayer assay for measuring the single-molecule membrane-binding behavior Ax647-PIP5KB at low and high membrane surface densities of PIP5KB. Note that the ‘high-density’ kinase organization does not imply a specific oligomerization state. (B) Single-molecule dwell times of Ax647-PIP5KB measured in the presence of increasing concentrations of unlabeled PIP5KB (0–50 nM). Ax647-PIP5K dwell time was calculated by fitting log₁₀(1 – cumulative distribution frequency [CDF]) to either a single- or double-exponential decay curve (black dashed lines). Bin size equals 50 ms. See Table 1 for statistics. (C) Representative step-size distributions measured in the presence of either 1 pM Ax647-PIP5KB (red), 1 pM Ax647-PIP5KB + 50 nM PIP5KB (green), or 1 pM Ax647-PIP4KB (blue). Dashed black line represents the curve fit used to calculate the diffusion coefficient (see ‘Materials and methods’). See Table 1 for statistics. (D) Representative trajectories showing the time-dependent movement of a single-membrane-bound Ax647-PIP5KB (1 pM) in the absence or presence of 5 nM dark PIP5KB. (**B–D**) Membrane composition: 98% DOPC, 2% PI(4,5)P₂. (E) Montage of images showing supported membranes with increasing densities of Atto655 lipids used to calibrate the molecular density of membrane-bound Ax647-PIP5KB. Membrane composition: 96% DOPC, 4% PI(4,5)P₂. (F) Ax647-PIP5KB binds cooperatively to membranes containing PI(4,5)P₂ lipids. The density of membrane-bound PIP5KB was measured in the presence of increasing solution concentrations of Ax647-PIP5KB on membranes containing 0.5, 1, 2, or 4% PI(4,5)P₂ lipids. Lines represent curve fit using concerted model for cooperativity (i.e., Hill equation). n_H is the Hill coefficient. Points are mean values (N = 15–20 fluorescent intensity measurements per sample from one technical replicate).

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Membrane-mediated dimerization of PIP5K

The ability of PIP5K proteins to associate with PI(4,5)P₂-containing membranes as monomers at low molecular densities (~0.01 PIP5K/µm²) confirms that PIP5K is predominantly monomeric when diluted to low solution concentrations. This supports a model that PIP5K exists in a weak monomer–dimer equilibrium in solution. However, membrane association reduces the effects of translational and rotational entropy, both of which oppose dimerization in solution, enabling even weakly dimerizing species in solution to robustly dimerize on membranes (Chung et al., 2018; Lin et al., 2014). Thus, we anticipated that membrane binding of PIP5K would favor dimerization at some threshold membrane surface density. In order to characterize PIP5K dimerization as a function of membrane surface density, we performed single-molecule-tracking experiments in the presence of low (~0.01 molecules/µm²) and high (~100 molecules/µm²) densities of membrane-bound PIP5KB (Figure 2A). These measurements reveal molecular binding dwell time on the membrane as well as diffusive mobility, both of which are affected by dimerization. For the single-molecule dwell time measurements, we switched to using the more photostable Alexa647 dye (referred to as Ax647) conjugated to either PIP4KB or PIP5KB (τ_bleach = 26.7 s, Figure 2—figure supplement 1). In the presence of a low density of noninteracting Ax647-PIP5KB monomers (i.e., 1–5 pM solution concentration), the distribution of single-molecule dwell times could be fit to a single exponential with a characteristic dwell time of ~0.45 s (Figure 2B, Figure 2—figure supplement 2, Figure 2—video 1). When we increased the solution concentration of dark unlabeled PIP5KB, we observed a second population of long dwelling Ax647-PIP5KB molecules (Figure 2B, Table 1, Figure 2—video 2). At concentrations greater than 1 nM dark unlabeled PIP5KB, the resulting dwell time distributions for Ax647-PIP5KB were best fit to a two-species model with two characteristic dwell times (Figure 2B, Table 1). In addition to observing an enhancement in the dwell time, we also observed a protein surface density-dependent decrease in the diffusion coefficient of membrane-bound Ax647-PIP5KB (Figure 2C, Table 1). Two-dimensional mobility on the membrane has previously been used as a highly effective measure of membrane surface dimerization reactions (Chung et al., 2018; Chung et al., 2019). A two-species model was required to fit the step-size distribution of membrane-bound Ax647-PIP5KB in the presence of high kinase density, which was similar to the step-size distribution of Ax647-PIP4KB measured at low molecular densities (Figure 2C). Examples of Ax647-PIP5KB molecules that transitioned between slow and fast diffusive states could also be seen when we inspected trajectories at an intermediate kinase density that favored PIP5K dimerization (Figure 2D).

Table 1

Protein density dependent changes in Ax647-PIP5KB membrane binding behavior.

Protein visualized	[5KB]	τ₁ ± SD (s)	τ₂± SD (s)	α ± SD	N	D₁ ± SD (µm²/s)	D₂ ± SD (µm²/s)	α ± SD	Steps
1 pM Ax647-5KB	0	0.453 ± 0.013	–	–	7018	0.148 ± 0.005	–	–	66,889
1 pM Ax647-5KB	0.2 nM	0.475 ± 0.037	–	–	4855	0.058 ± 0.011	0.194 ± 0.012	0.20 ± 0.06	48,888
1 pM Ax647-5KB	1 nM	0.559 ± 0.009	–	–	4783	0.038 ± 0.001	0.189 ± 0.004	0.27 ± 0.02	56,027
1 pM Ax647-5KB	5 nM	0.599 ± 0.055	1.354 ± 0.32	0.72 ± 0.17	4268	0.038 ± 0.002	0.173 ± 0.007	0.40 ± 0.04	66,680
1 pM Ax647-5KB	10 nM	0.895 ± 0.032	2.791 ± 0.27	0.94 ± 0.03	2362	0.039 ± 0.004	0.164 ± 0.002	0.48 ± 0.05	47,103
1 pM Ax647-5KB	50 nM	0.787 ± 0.057	2.08 ± 0.078	0.70 ± 0.03	1530	0.043 ± 0.002	0.142 ± 0.008	0.63 ± 0.03	35,678

1 pM Ax647-4KB	0					0.021 ± 0.005	0.058 ± 0.007	0.39 ± 0.15	227,275

SD, standard deviation from 3 to 5 technical replicates; N, total number of molecules tracked in 3–5 technical replicates; steps, total number of particle displacements measured in 3–5 technical replicates; alpha, fraction of molecules that have the characteristic dwell time or diffusion coefficient (τ₁ or D1), τ_bleaching, Ax647-5KB = 26.7 s (see Figure 2—figure supplement 1); membrane composition, 98% DOPC, 2% PI(4,5)P₂.

To quantify how membrane binding of PIP5KB changes as a function of the PI(4,5)P₂ lipid density and the kinase solution concentration, we used a method previously established by Galush et al., 2008 to measure the membrane surface density of Ax647-PIP5KB compared to a fluorescent lipid standard. Using defined molar concentrations of Atto655-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Atto655-DPPE) lipids incorporated into supported membranes, we calibrated the fluorescence intensity in order to measure the surface density of membrane-bound Ax647-PIP5KB (Figure 2E, Figure 2—figure supplement 3). This approach overcame the challenges associated with using fluorescence correlation spectroscopy (FCS) to quantify the high membrane surface density of slow-diffusing Ax647-PIP5KB. These measurements revealed two nonlinear membrane-binding behaviors of Ax647-PIP5KB. First, the density of membrane-bound Ax647-PIP5KB dramatically increased as a function of the PI(4,5)P₂ density (Figure 2F). Second, increasing the solution concentration promoted cooperative membrane binding of Ax647-PIP5KB based on elevated protein densities (Figure 2F). Fitting the membrane-binding curves with a concerted model for cooperativity yielded dissociation constants of 212 nM and 52 nM for Ax647-PIP5KB in the presence of 2% and 4% PI(4,5)P₂ lipids, respectively (Figure 2F). To determine whether the density-dependent change in the dwell time and the step-size distributions of Ax647-PIP5KB were dependent on dimerization of the kinase domain, we sought to characterize a mutant that disrupts the PIP5KB dimer interface.

Inspection of the zPIP5KA crystal structure (Hu et al., 2015) and primary amino acid sequence alignment revealed a high degree of conservation between PIP5K homologs and paralogs (Figure 3A and B). Based on conservation of the primary amino acid sequence, we mutated the dimer interface of human PIP5KB to make the kinase constitutively monomeric. Using smTIRF microscopy, we compared the dwell times and diffusion coefficients of Ax647-PIP5KB and Ax647-PIP5KB (D51R) under conditions with either low and high protein densities on SLBs. When measured at a low protein surface density (~0.01 molecule/µm²), the PIP5KB (D51R) mutant still bound cooperatively to PI(4,5)P₂ lipids in a manner that was indistinguishable from the wild-type kinase (Figure 3C and D, Table 2). Under these conditions, the diffusion coefficients of membrane-bound Ax647-PIP5KB and Ax647-PIP5KB (D51R) were indistinguishable (Figure 3—figure supplement 1). In contrast, single-molecule membrane-binding experiments performed using a high kinase surface density (~100 molecules/µm²) revealed that the single-molecule dwell time of Ax647-PIP5KB increased, while the dwell time of the D51R mutant remained unchanged in the presence of 50 nM unlabeled PIP5KB (Figure 3E, Table 2). In addition, the diffusion coefficient of wild-type Ax647-PIP5KB decreased due to membrane-mediated dimerization, while diffusivity of Ax647-PIP5KB (D51R) remained unchanged in the presence of 50 nM dark PIP5KB (Figure 3F).

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PIP5K binds cooperatively to PI(4,5)P₂ independent of dimerization.

(A) Kinase domain orientation for the zebrafish PIP5KA homodimer (4TZ7.pdb). Salt bridges formed between Asp and Arg side chains are colored and shown in zoomed images. (B) Sequence alignment between zPIP5KA, human PIP5KA, human PIP5KB, and human PIP5KC highlights conservation of residues in dimer interface. (**C, D**) Dimerization is not required for cooperative PI(4,5)P₂ binding. Representative single-molecule dwell time distributions measured at low protein density in the presence of either (C) 1–5 pM Ax647-PIP5KB and (D) 1–5 pM Ax647-PIP5KB (D51R). Membrane composition: 1, 2, or 4% PI(4,5)P₂, plus 96–99% DOPC. (**E, F**) Single-molecule dwell times and step-size distributions measured at ‘high density’ in the presence of 50 nM non-fluorescent PIP5KB. (E) High protein surface density of PIP5KB increases the dwell time of Ax647-PIP5KB, but not Ax647-PIP5KB (D51R). (F) Membrane-mediated dimerization is responsible for the protein density-dependent decrease in Ax647-PIP5KB diffusion coefficient. (**C–F**) Membrane composition: 96% DOPC, 4% PI(4,5)P₂. Dashed line represents fits of the dwell time distributions. See Table 2 for statistics.

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Table 2

Dimerization dependent changes in Ax647-PIP5KB membrane binding behavior.

Protein visualized	[5KB]	% PIP₂	τ₁ ± SD (s)	τ₂ ± SD (s)	α ± SD	N	D₁ ± SD (µm²/s)	D₂ ± SD (µm²/s)	α ± SD	Steps
25 pM Ax647-5KB	0	1	0.096 ± 0.05	–	–	3422	–	–	–	–
5 pM Ax647-5KB	0	2	0.64 ± 0.092	–	–	4525	0.171 ± 0.006	–	–	174,748
2 pM Ax647-5KB	0	4	0.74 ± 0.088	3.43 ± 0.29	0.59 ± 0.06	1920	–	–	–	–
25 pM Ax647-5KB (D51R)	0	1	0.090 ± 0.01	–	–	4848	–	–	–	–
5 pM Ax647-5KB (D51R)	0	2	0.649 ± 0.043	–	–	2542	0.174 ± 0.001	–	–	238,548
2 pM Ax647-5KB (D51R)	0	4	0.864 ± 0.077	3.97 ± 0.37	0.58 ± 0.03	1850	–	–	–	–
1 pM Ax647-5KB	50 nM	2	0.709 ± 0.24	2.49 ± 0.27	0.53 ± 0.08	9693	0.022 ± 0.002	0.067 ± 0.005	0.50 ± 0.07	311,198
1 pM Ax647-5KB (D15R)	50 nM	2	0.416 ± 0.02	–	–	8218	0.133 ± 0.008	–	–	74,928

SD, standard deviation from 3 to 5 technical replicates; N, total number of molecules tracked in 3–5 technical replicates; steps, total number of particle displacements measured in 3–5 technical replicates; alpha, fraction of molecules that have the characteristic dwell time or diffusion coefficient (τ₁ or D₁), τ_bleaching, Ax647-5KB = 26.7 s (see Figure 2—figure supplement 1); membrane composition, 96–99% DOPC, 1–4% PI(4,5)P₂.

Having established that the density-dependent changes in Ax647-PIP5KB membrane-binding behavior are mediated by dimerization, we sought to establish conditions to directly visualize dimerization of membrane-bound PIP5KB with single-molecule resolution. For these experiments, we recombinantly expressed and purified mNG-PIP5KB and mNG-PIP5KB (D51R). Based on the SEC elution profiles, mNG-PIP5KB and mNG-PIP5KB (D51R) adopted distinct oligomerization states when loaded at a solution concentration of 50 µM (Figure 4A). This concentration is approximately 1000× greater compared to the reported cellular concentration of PIP5K (Hein et al., 2015). Next, we performed single-molecule imaging to compare the molecular brightness of mNG-PIP5KB and mNG-PIP5KB (D51R) immediately following attachment to membranes containing 4% PI(4,5)P₂ lipids (Figure 4B). In the presence of 100 pM solution concentration, the molecular brightness distributions of mNG-PIP5KB and mNG-PIP5KB (D51R) were indistinguishable (Figure 4C and D). Under these conditions, both kinases in solution associated with membranes exclusively as monomers. Next, we compared the fluorescence intensity of mNG-PIP5KB and mNG-PIP5KB (D51R) already membrane bound and potentially associated with other membrane-bound kinases. For this analysis, we compared the molecular brightness distributions mNG-PIP5KB and mNG-PIP5KB (D51R) at identical membrane surface densities of ~600 mNG-labeled kinases per field of view (i.e., 0.2 kinases/µm²). Under these conditions, we observed two classes of particles with distinct fluorescence intensities (Figure 4E and F). Compared to mNG-PIP5KB (D51R), there was a fivefold higher frequency of observing mNG-PIP5KB dimers (Figure 4G and H). Photobleaching analysis of the bright mNG-PIP5KB particles revealed a stepwise decrease in fluorescence consistent with a dimer (Figure 4I). In our cell lysate experiments, we also observed similar differences in molecular brightness comparing mNG-PIP5KA and mNG-zPIP5KA to their respective dimer interface mutants, mNG-PIP5KA (D92R) and mNG-zPIP5KA (D84R) (Figure 4—figure supplement 1).

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Direct visualization of mNG-PIP5KB membrane-mediated dimerization.

(A) Size-exclusion chromatography (SEC) elution profiles of purified mNG-PIP5KB and mNG-PIP5KB (D51R). The monomeric molecular weight of each kinase equals 75 kDa. Protein was at a concentration of 50 µM (~2 mg total) loaded on a Superdex 200 column. Based on the A280, peak elution concentration is 10–20 µM. (B) Diagram showing the experimental setup for visualizing membrane binding of mNG-PIP5KB (WT and D51R). (**C–, D**) Molecular brightness distributions of mNG-PIP5KB and mNG-PIP5KB (D51R) based on brightness of single kinases first attaching to the supported membrane (N = 2424 total binding events, four technical replicates in C; N = 1747 total binding events, four technical replicates in D). (**E–, F**) Molecular brightness distributions based on equilibrium membrane binding of either mNG-PIP5KB or mNG-PIP5KB (D51R). Molecules with a threshold particle intensity ≥8200 (A.U.) were considered dimeric. Values printed above double arrows equal percentage of apparent dimers (N = 11,355 mNG-PIP5KB particles in E; N = 12,759 mNG-PIP5KB (D51R) particles in F). (G) Image showing the localization of membrane-bound mNG-PIP5KB observed in the presence of a 100 pM solution concentration. Arrows indicate mNG-PIP5KB dimers. (H) Intensity line scan through line draw in (G). (I) Stepwise photobleaching of a membrane-bound mNG-PIP5KB dimer. (J) Direct visualizing of membrane-mediated dimerization in the presence of mNG-PIP5KB. (**C–J**) Data collected in the presence of 100 pM mNG-PIP5KB or mNG-PIP5KB (D51R). Membrane composition: 96% DOPC, 4% PI(4,5)P₂.

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To directly visualize the association and dissociation dynamics of mNG-PIP5KB dimers, we tracked membrane-bound kinases with high temporal resolution. Under these imaging conditions, we observed a small fraction membrane-bound mNG-PIP5KB monomer diffuse and merge together, forming dimers that persist for 1–2 s before photobleaching or dissociating back into monomers (Figure 4J, Figure 4—video 1). These dimerization dynamics were not observed for mNG-PIP5KB (D51R). Considering the high membrane surface density used for these experiments, the small fraction of apparent mNG-PIP5KB (D51R) ‘dimers’ (i.e., 0.9%) represents kinases that transiently cross paths on the membrane without forming a stable dimer complex. Overall, these results support a model in which mNG-PIP5KB can exist as a monomer in solution. However, membrane binding and lateral diffusion promotes membrane-mediated dimerization in a density-dependent manner.

Dimerization potentiates PIP5K activity independent of enhancing membrane avidity

Dimerization of zPIP5KA was previously shown to be required for lipid kinase activity in vitro (Hu et al., 2015). Comparing the kinetics of PI(4,5)P₂ production on SLBs in the presence of either PIP5KB and PIP5KB (D51R) revealed that dimerization enhances catalytic activity, but is not essential for generation of PI(4,5)P₂ (Figure 5A and B). A similar reduction in catalytic activity was observed for mutations that disrupt dimerization of human PIP5KA (Figure 5—figure supplement 1). Previous characterization of a dimerization-deficient zPIP5KA, D84R, using circular dichroism demonstrated that disruption of the dimerization interface does not alter protein folding (Hu et al., 2015). To confirm that the PIP5KB dimer interface mutation, D51R, did not suffer from protein misfolding, we introduced an R254D compensatory mutation homologous to the zPIP5KA (R287D) mutation previously shown to restore the zPIP5KA dimer interface mutation, D84R (Figure 3B; Hu et al., 2015). Kinetic analysis of PIP5KB (D51R/R254D)-driven lipid phosphorylation showed that restoring the dimer interface through this orthogonal salt bridge produced a kinase with catalytic activity indistinguishable from wild-type PIP5KB (Figure 5C and D).

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Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

(**A–C**) Representative kinetic traces monitoring the production of PI(4,5)P₂ in the presence of 1–5 nM PIP5KB, PIP5KB (D51R), or PIP5KB (D51R/R254D). The PI(4,5)P₂ membrane surface density was estimated based on the membrane localization of 20 nM Ax488-PLCδ. Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Quantification of reaction half-time from trajectories shown in (**A–C**). Bars equal mean values (N = 2–4 reactions per concentration, error = SD). (E) Equilibrium diagram showing the relationship between PIP5KB membrane binding, oligomerization, and catalysis. (F) Equilibrium fluorescence intensity of membrane-bound mNG-PIP5KB and mNG-PIP5KB (D51R) measured at varying solution concentrations. Points are mean values (N = 15–20 fluorescent intensity measurements per sample from one experiment). Membrane composition: 96% DOPC, 4% PI(4,5)P₂. (G) Membrane-mediated dimerization enhances PIP5KB catalytic efficiency independent of membrane localization. Membrane localization of mNG-PIP5KB (WT and D51R) and production of PI(4,5)P₂ were simultaneously monitored on supported membranes. Each data point in the plot represents the instantaneous velocity expressed as the number of PI(4,5)P₂ lipids generated per µm² per second per kinase as a function of the substrate density. The reaction catalyzed by PIP5KB displays the following three phases: (1) slow kinetics at reaction start, (2) rise to maximum reaction velocity, and (3) gradual decline in reaction velocity due to substrate depletion. (H) Feedback profiles for PIP5KB (WT and D51R). The following equations were used for curve fitting: ${(k}_{0}^{+} + k_{1}^{+} x + k_{2}^{+} x^{2}) (1 - x)$ for PIP5KB and ${(k}_{0}^{+} + k_{1}^{+} x) (1 - x)$ for PIP5KB (D51R) where $x$ represents the phosphatidylinositol phosphate (PIP) lipid composition.

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To determine whether the differences in catalytic activity of the dimer mutant were caused by a reduction in PIP5KB (D51R) membrane recruitment or dimerization-induced change in catalytic efficiency, we compared the relative membrane binding strength of mNG-PIP5KB and mNG-PIP5KB (D51R). Over a broad range of solution concentrations, we observed approximately twofold higher membrane density for mNG-PIP5KB, compared to mNG-PIP5KB (D51R) (Figure 5F). This suggests that the reduction in membrane binding affinity is not the primary reason for the sevenfold reduction in activity observed when we disrupt the dimer interface of PIP5K.

Next, we simultaneously measured the kinetics of lipid phosphorylation and quantified the absolute density of membrane bound mNG-PIP5K. To determine whether dimerization enhances the catalytic efficiency of PIP5K, we calculated the effective phosphorylation rate constants per enzyme based on the calibrated surface density of mNG-PIP5KB (Figure 5—figure supplement 2). Compared to typical solution Michaelis−Menten kinetics, the density of membrane-bound mNG-PIP5KB changes over the course of the lipid phosphorylation reactions described in Figure 5E. To account for this change, the effective phosphorylation rate per membrane-bound PIP5KB molecule, $v_{m o l e c u l e} (t)$ , was calculated using the following equation:

v_{m o l e c u l e} (t) = \frac{d P (t)}{d t} \frac{1}{E (t)}

where E is the surface density of mNG-PIP5KB, and P is the surface density of PI(4,5)P₂ on the membrane at any time during the reaction. The PI(4)P density at each time point, $S (t)$ , was approximated by subtracting $P (t)$ from the initial PIP lipid density, $S_{0}$ :

S (t) = S_{0} - P (t)

By plotting $v_{m o l e c u l e} (t)$ against the substrate density, $S (t)$ , we obtained a Michaelis–Menten plot with a slope equal to the effective phosphorylation rate constant per kinase (Figure 5G). The calculated phosphorylation rate constant for the dimer mutant, PIP5KB (D51R), had a single rate constant of 2.3 × 10^–4 lipids/µm²•s per enzyme throughout the observed reaction trajectory. Conversely, the phosphorylation rate constant for wild-type PIP5KB began with a slow rate and then transitioned to a rate of 2.7 × 10^–3 lipids/µm²•s per kinase as the reaction progressed (Figure 5G, see arrows indicating the transition from slow to fast kinetics for wild-type PIP5KB). After the reaction reached the maximum velocity, the kinetics gradually slowed down due to substrate depletion. Overall, the difference in per-molecule reaction kinetics comparing PIP5KB and PIP5KB (D51R) was consistent with dimerization enhancing lipid kinase activity (Figure 5E) and establishes a positive feedback mechanism.

Comparing the shape of the kinetic traces for PIP5KB and PIP5KB (D51R) also revealed striking differences in the complexity of their positive feedback loops. To analyze the feedback profiles, we plotted the rate of PI(4,5)P₂ production, $d x / d t$ , as a function of the reaction coordinate, x, as $x \equiv σ P I (4, 5) P 2 / (σ P I (4, 5) P 2 + σ P I (4) P)$ . $σ$ denotes the membrane density of each PIP lipid species throughout the entire reaction trajectory. The overall reaction rate can be expressed as

\frac{d x}{d t} = k (x) ∙ (1 - x)

where $k (x)$ is a function that characterizes the reaction rate, including any dependence on the instantaneous PIP lipid composition ( $x$ ). As previously described by Hansen et al., 2019, the overall rate constant, $k (x)$ , can be expressed as a power series, $k (x) = k_{0} + k_{1} x + k_{2} x^{2} + \dots$ . This provides a convenient way of examining the type of feedback; the order of feedback is revealed by the $x$ -dependence of $k (x)$ . Plotting the derivative of the PIP5KB kinase reaction traces against the reaction coordinate (i.e., PIP lipid composition), $x$ , the curve displayed a high degree of asymmetry that required a second-order term to fit ( $k_{W T} (x) = k_{0} + k_{1} x + k_{2} x^{2}$ ) (Figure 5H). In contrast, the $d x / d t$ curve for the PIP5KB (D51R) dimer mutant was parabolic and could be fit using an equation that describes an enzyme with simple first-order positive feedback ( $k_{D 51 R} (x) = k_{0} + k_{1} x$ ) based on product binding (Figure 5H). In previous studies, PIP5KB was shown to exhibit higher-order positive feedback (Hansen et al., 2019). One potential source of higher-order positive feedback is through the ability of PIP5K to bind to multiple PI(4,5)P₂ lipids. However, mapping the feedback strength based on the reaction coordinate revealed that dimerization is predominantly responsible for PIP5K higher-order positive feedback. Together, these results provide strong evidence that dimerization enhances the activity of PIP5K by both enhancing the membrane avidity and potentiating lipid kinase activity through a mechanism that is consistent with allosteric regulation.

PIP5K dimerization increases the stability of PIP compositional patterns

The PIP5KB positive feedback mechanism previously enabled us to reconstitute a bistable lipid kinase–phosphatase competitive reaction that breaks symmetry and produces PI(4)P and PI(4,5)P₂ compositional patterns on supported membranes (Figure 6A; Hansen et al., 2019). Based on the ability of PIP5KB to undergo membrane-mediated dimerization and the corresponding nonlinear positive feedback, we hypothesized the kinetic bistability in this system could be dependent on membrane-mediated dimerization. To test this hypothesis, we compared the ability of PIP5KB and the PIP5KB (D51R) dimer interface mutant to form bistable PIP compositional patterns in the presence of an opposing 5-phosphatase with engineering positive feedback based. This was achieved by fusing a minimal PI(4)P binding motif, referred to as DrrA (or SidM/DrrA), to OCRL (Hammond et al., 2014; Zhu et al., 2010). The resulting chimeric 5-phosphatase, DrrA-OCRL, was previously shown to exhibit positive feedback based on PI(4)P recognition (Hansen et al., 2019). In the presence of 50 nM PIP5KB (WT or D51R) and 30 nM DrrA-OCRL, we found that the activity of the D51R dimer mutant was strongly impaired compared to wild-type PIP5KB (Figure 6B). Restoring the PIP5KB dimer interface with a charge reversal mutation, D51R/R254D, allowed us to reconstitute bistable compositional patterns that were indistinguishable from those formed in the presence of wild-type PIP5K (Figure 6B). Again, the behavior of PIP5KB (D51R/R254D) indicated that the D51R mutation does not structurally harm the kinase. Taking into consideration the weakened positive feedback of the dimer mutant, we raised the solution concentration of PIP5KB (D51R) until we were able to balance the opposing 5-phosphatase activity. Using a 20-fold higher concentration of PIP5KB (D51R), compared to wild-type, we identified a concentration regime that allowed us to reconstitute PIP compositional pattern formation on supported membranes (Figure 6B). During the early stages of pattern formation, the surface area and morphology looked very similar to the compositional patterns reconstituted in the presence of wild-type PIP5KB. However, after a couple of minutes the PI(4,5)P₂ compositional patterns generated by PIP5KB (D51R) were consumed by the surrounding 5-phosphatase dominated reaction (Figure 6B).

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PIP5K dimerization stabilizes phosphatidylinositol phosphate (PIP) compositional patterns.

(A) Diagram showing the network architecture of the bistable kinase–phosphatase-competitive reaction. The positive feedback loop of the 5-phosphatase, DrrA-OCRL, is regulated by product binding (Hansen et al., 2019). Note that DrrA is also commonly known as SidM/DrrA in *L. pneomophila*. (B) PIP5KB dimerization increases robustness of PIP compositional pattern formation. Montage image sequences showing the formation PIP compositional patterns in the presence 50 nM PIP5KB (WT, D51R, or D51R/R254D, montage rows 1–3) or 1 µM PIP5KB (D51R, montage row 4). (C) Localization of mNG-PIP5KB (WT or D51R) during PIP compositional pattern formation. Reactions were reconstitution in the presence of either 50 nM mNG-PIP5KB or 1 µM mNG-PIP5KB (D51R). (D) Plot showing the change in mNG-PIP5KB membrane intensity compositional patterns enriched in PI(4,5)P₂ lipids. The dashed line represents the mean fluorescence intensity from N = 10 membrane domains. (**B–D**) All reactions contain the specified concentration of PIP5KB (WT or mutants), plus 20 nM DrrA-OCRL, 20 nM Ax488-PLCδ, and 20 nM Ax647-DrrA. Initial membrane composition: 96% DOPC, 2% PI(4)P, 2% PI(4,5)P₂.

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To determine whether the inability of monomeric PIP5KB (D51R) to form stable compositional patterns was due to a relatively lower membrane surface density of the dimer mutant, we compared the membrane localization dynamics of mNG-PIP5KB and mNG-PIP5K (D51R) during the formation of PIP compositional patterns (Figure 6C). In the presence of mNG-PIP5KB, the kinase membrane density steadily increased and then plateaued shortly after pattern formation (Figure 6D, Figure 6—video 1). In contrast, the membrane surface density of mNG-PIP5K (D51R) sharply increased immediately after initiating the kinase–phosphatase competitive reactions, reaching a density nearly two times greater compared to the steady-state density observed for mNG-PIP5KB (Figure 6D, Figure 6—video 2). After the mNG-PIP5KB (D51R)-driven reaction reached a peak surface density that was sufficient to generate the bistable PIP compositional patterns, the PI(4,5)P₂-containing membrane domains rapidly decreased in size and disappeared (Figure 6D). Overall, mNG-PIP5K (D51R) reached a sufficiently high membrane density to form compositional patterns, but lacked the catalytic activity needed to oppose the competing 5-phosphatase reaction and maintain the established PI(4,5)P₂ compositional pattern.

Reaction trajectory variation is enhanced by membrane-mediated dimerization

We previously reported that PIP5K-dependent lipid phosphorylation reactions exhibit a high degree of reaction trajectory variation when reconstituted on SLBs that are partitioned into micron-length-scale membrane corrals (Hansen et al., 2019). Based on the coupling induced by membrane-mediated dimerization, we hypothesized that the dimerization could provide the molecular basis for the previously observed enhanced reaction trajectory variation. To measure dimerization-dependent differences in reaction trajectory variation, we microfabricated an array of 5 µm × 5 µm chromium barrier onto the underlying glass coverslip. This approach allowed us to visualize hundreds of identical membrane reactions in parallel that continuously exchange with the surrounding solution environment. Under these conditions, PIP5KB reaction trajectories displayed a high degree of kinetic heterogeneity (Figure 7A and B, Figure 7—video 1). While the time to finish was highly heterogeneous, the reaction rate of the initial and later part of the reaction was quite homogeneous. In contrast, reactions reconstituted in the presence of PIP5KB (D51R) showed little heterogeneity, confirming that dimerization strongly enhanced variation in corral reaction trajectory (Figure 7B, Figure 7—video 2). Overall, the observed reaction heterogeneity was primarily driven at higher product concentrations, where higher-order positive feedback dominates. Formation of the PIP5K dimer creates a highly active kinase with a strengthened positive feedback and faster reaction velocity.

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Reaction trajectory variation based on stochastic membrane-mediated dimerization.

(A) Lipid phosphorylation reactions reconstituted in 5 µm × 5 µm chromium-patterned-supported membranes in the presence of 5 nM PIP5K and 10 nM PIP5K (D51R). Reactions were visualized using 20 nM Alexa488-PLCδ. Initial membrane composition: 96% DOPC, 4% PI(4)P. (B) Reaction trajectories plots from (A) from N = 100 membrane corrals. The mean reaction trajectory is plotted in orange. (C) Time course of bistable kinase–phosphatase reaction reconstituted in 5 µm × 5 µm corrals in the presence of 50 nM PIP5K, 30 nM DrrA-OCRL, 20 nM Alexa488-PLCδ, and 20 nM Ax647-DrrA. (D) Dimerization enhances the ability of PIP5K to win competitive bistable kinase–phosphatase reaction in the presence of OCRL. Representative steady-state images showing outcomes reconstituted in the presence of 3 µM OCRL, plus either 18 nM PIP5KB or 370 nM PIP5KB (D51R). The concentration of PIP5KB (WT and D51R) used for these experiments was determined based on achieving an approximately 50–50 reaction outcome in the presence of 3 µM OCRL. (E) Quantification of final reaction outcome in (D) from a single experiment (N > 100 corrals per OCRL concentration). (**C–E**) Initial membrane composition: 96% DOPC, 2% PI(4)P, and 2% PI(4,5)P₂.

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Stochastic effects can enhance bistability

Nonlinear positive feedback is sufficient to establish bistability in a competitive reaction, even on scales over which stochastic variations average to essentially zero. The stable patterns seen in Figure 6B (wild-type and D51R/R254D) are likely a representation of this non-stochastic bistability, with two intrinsically stable steady states. The dimer mutant, however, shows only linear positive feedback (see Figure 5H) but still shows at least transient bistability in Figure 6B–D. This is a manifestation of stochastic bistability, referring to bistable behavior in systems that inherently lack two stable steady states (Bishop and Qian, 2010; Hansen et al., 2019; Artyomov et al., 2007; To and Maheshri, 2010). For the wild-type PIP5KB, dimerization enhances stochastic variation in reaction rate and thus we hypothesize will expand the range of conditions over which bistable behavior is possible, even spanning beyond the boundaries of intrinsic bistability. To test this hypothesis, we examine competitive reactions with OCRL phosphatase for PIP5KB and PIP5KB (D51R) under geometric confinement, where stochastic effects are prominent. Reconstitution of the PIP5KB and OCRL competitive reactions in 5 μm × 5 μm membrane corrals reveals strong bistability, reaching final membrane compositions in each corral consisting of steady states dominated by either PI(4)P or PI(4,5)P₂ composition (Figure 7C). To compare with the dimer mutant, we titrated the competing OCRL concentration against fixed concentrations of either PIP5KB and PIP5KB (D51R) and examine the resultant bistability (Figure 7D). Wild-type PIP5KB exhibits a much more robust bistability, which spans a substantially wider range of competing phosphatase concentrations than observed for the PIP5KB (D51R) dimer mutant. Quantification of this effect is plotted in Figure 7E. Dimerization of PIP5K thus ensures that the kinase–phosphatase-competitive reactions can achieve a bistable response over a broad range of opposing 5-phosphatase activity.

Discussion

Dimerization as a mechanism for potentiating PIP5K activity

Cooperative PI(4,5)P₂ binding and membrane-mediated dimerization provide synergistic mechanisms to increase the rate of PI(4,5)P₂ production through the enhanced localization and increased catalytic efficiency of PIP5K (Figure 8). Supporting a mechanism of allosteric regulation, our results indicate that dimerization enhances PIP5K lipid kinase activity by directly increasing k_cat. This effect is independent of increasing membrane avidity of PIP5K. Due to a lack of structural biochemistry, there is currently a gap in knowledge concerning the role dimerization, PIP lipid binding, and the nucleotide state serve in regulating conformational states of PIP5K. Given our limited structural understanding of PIP5K (Liu et al., 2016; Hu et al., 2015; Muftuoglu et al., 2016), some researchers have used molecular dynamic simulations to elucidate how membrane docking of PIP5K is controlled (Amos et al., 2019). Working under the assumption that PIP5K is constitutively dimeric, Amos et al. reported that only a single kinase domain can engage substrate when the dimer is docked on a PI(4)P-containing membrane. Consistent with this observation, we find that the dimerization modestly enhanced membrane binding. If this mechanism is accurate, membrane-mediated dimerization is expected to enhance processivity of membrane-bound PIP5K by allowing kinase domains to toggle between states of catalysis and membrane binding. This mechanism would ensure that dimeric PIP5K remains bound to the membrane during enzyme catalysis, while the monomeric PIP5K would likely catalyze a single PI(4)P phosphorylation reaction before dissociating. According to our single-molecule biophysical studies, we believe that both subunits of the PIP5K kinase domain can engage the membrane. This is based on the enhanced dwell time and slower diffusion coefficient observed when Ax647-PIP5K is visualized is high protein density (i.e., ~100 molecules/µm²). Incorporating reversible dimerization, catalysis, and PI(4,5)P₂ binding into future molecular dynamic simulations could provide new insight into the mechanism of membrane docking.

Figure 8

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Model for PIP5K membrane-mediated dimerization.

Mediated by electrostatic interactions, PIP5K can associate with membranes as a monomer. The specificity loop stabilizes membrane association and enables PIP5K to catalyze the phosphorylation of PI(4)P to generate PI(4,5)P₂. Increasing densities of PI(4,5)P₂ and membrane-bound PIP5K promote membrane-mediated dimerization, which leads to enhanced catalytic efficiency.

PIP5K dimerization increases reaction trajectory variation

Cooperative PI(4,5)P₂ binding, membrane-mediated dimerization, and low molecular copy number of PIP5K provide several mechanisms for enhancing stochasticity in membrane-associated lipid phosphorylation reactions. Previously, we reported that reaction velocity fluctuations driven by stochastic binding and unbinding of PIP5K on the membrane could drive the system to a predominantly PI(4,5)P₂ state, even in the presence of high levels of opposing lipid phosphatase activity that were sufficient to drive the system predominantly to the PI(4)P state in bulk (Hansen et al., 2019). The resulting emergent property is that the reaction outcome depends on system size, which we termed stochastic geometry sensing; related scale-dependent phenomena have been called reaction inversion in some theoretical work (Ramaswamy et al., 2012). Here, we report that dimerization is responsible for the higher-order positive feedback exhibited by PIP5K, and this contributes to the robustness with which this system exhibits stochastic geometry sensing. Without dimerization, PIP5K displays a simple linear positive feedback profile. Comparing the lipid phosphorylation reaction trajectory variation of PIP5KB and PIP5KB (D51R) highlights how dimerization enhances stochasticity of lipid kinase phosphorylation reactions and will thus amplify any effects dependent on stochastic fluctuations. Given the broad dynamic range of kinase densities and activities that can emerge from these biochemical mechanisms, stochasticity in PIP5K signaling has the potential to strongly influence signaling events orchestrated at the plasma membrane in living cells.

PIP5K localization and lipid kinase activity in cells

The strong lipid phosphorylation activity of PIP5K observed in vitro raises questions about the steady-state localization and activity of PIP5K in cells. Although new molecular mechanisms concerning PIP5K activation have been revealed through single-molecule characterization of PIP5K in vitro, it remains challenging to interpret how dimerization, PI(4,5)P₂ binding, and interactions with peripheral membrane proteins regulate membrane localization of PIP5K in vivo. Complicating our interpretation of cellular localization, PIP5K can also reportedly interact with phosphatidylserine and sterol lipids, which modulate lipid kinase activity (Nishimura et al., 2019). In addition, PIP5KA, PIP5KB, and PIP5KC paralogs can potentially form heterodimeric complexes that have unique membrane avidities and kinase activities (Lacalle et al., 2007).

Human proteomic data has estimated the cellular concentration of PIP5K in mammalian cells to be 10–20 nM (Hein et al., 2015). Based on the weak monomer–dimer equilibrium, PIP5K is predicted to exist predominantly as a monomer in the cytoplasm. However, receptor activation could shift the steady-state localization of PIP5K to favor membrane-dependent dimerization and enhanced lipid kinase activity. This could be achieved by increasing PIP5K membrane recruitment through interactions with receptors, endocytic machinery, or small GTPases (Halstead et al., 2010; Honda et al., 1999; Funakoshi et al., 2010). If the steady-state localization of PIP5K exists near a threshold for activation, a minor enhancement in localization could promote PIP5K dimerization and trigger the PI(4,5)P₂-dependent positive feedback loop. Future biochemical characterization of PIP5K will enable researchers to generate better separation of function mutants that allow the functional significance of specific protein–protein and protein–lipid interactions to be studied in the context of cell signaling. Determining how cells regulate the strength and duration of PIP5K lipid phosphorylation reactions during receptor activation will also help elucidate the role cooperative PI(4,5)P₂ binding and dimerization serve in enhancing PIP5K-dependent production of PI(4,5)P₂ during cell signaling.

One challenge in establishing an accurate model for PIP5K membrane binding and catalysis is the gap in knowledge concerning how PIP5K binds to PI(4,5)P₂ and other lipids. Previously published cell biology data reported that several basic residues are critical for PIP binding and plasma membrane localization in cells (Fairn et al., 2009). However, these basic residues are dispensable for PIP5K membrane binding and activity in vitro (Hansen et al., 2019). Looking at the crystal structure of zPIP5KA (Hu et al., 2015; Muftuoglu et al., 2016), there are several motifs of unresolved electron density and domains extending beyond the N- and C-terminus of the kinase domain for which we lack high-resolution structural data. Defining the functional relevance of those domains in controlling PIP5K membrane association could provide new insight into the molecular basis of PI(4,5)P₂-binding specificity. Considering the cooperative nature of the PIP5K and PI(4,5)P₂ interaction, visualization of fluorescently labeled PIP5K in cells could be used to infer local membrane density of PI(4,5)P₂ at the plasma membrane based on single-molecule dwell times of PIP5K. We know from previous work that PIP5K becomes enriched at synaptic vesicles (Nakano-Kobayashi et al., 2007) and focal adhesions (Ling et al., 2002; Di Paolo et al., 2002). However, it remains unclear whether localization of PIP5K to these cellular structure requires interactions with PI(4,5)P₂ lipids, dimerization of the kinase domain, or recruitment by peripheral membrane proteins.

Negative regulation of PIP5K signaling

The strong positive feedback loop displayed by PIP5K raises questions concerning how lipid kinase activity is inhibited once production of PI(4,5)P₂ is accelerated. Left unregulated, the PIP5K-positive feedback loop has the potential to generate excessively high concentrations of PI(4,5)P₂ in cells, which would be detrimental to numerous signaling pathways that rely on cellular PIP lipid homeostasis. New evidence suggests that, in vivo and in vitro, PIP4K can attenuate PIP5K activity through the formation of a membrane-bound hetero-kinase complex (Wang et al., 2019; Wills et al., 2022). Deciphering the molecular basis of PIP4K-PIP5K complex formation using single-molecule in vitro measurements will be critical for determining both the mechanism of kinase inhibition and for generating separation of functions mutants that perturb this regulatory mechanism.

By limiting the concentration of freely available PI(4,5)P₂, cells can potentially control the strength and duration of the PIP5K-positive feedback loops. In vitro, PIP5K interacts strongly with supported membranes containing 2–4% PI(4,5)P₂. Although the total concentration of PI(4,5)P₂ is estimated to be 0.5–5% in the plasma membrane (Wenk et al., 2003; Mitchell et al., 1986; Nasuhoglu et al., 2002), the concentration of freely available PI(4,5)P₂ is potentially lower. PI(4,5)P₂-sequestering proteins, like MARCKS, are present at micromolar concentrations in cells (Brudvig and Weimer, 2015) and likely limit the concentration of freely available PI(4,5)P₂ at the plasma membrane. Lipid phosphatases, phosphatidylinositol-3 kinase (PI3K), and phospholipases also regulate the conversion of PI(4,5)P₂ into other lipid species and secondary metabolites (Balla, 2013). In the case of early phagocytosis, macrophages can put the brakes on PIP5K activity by activating phospholipase C, which cleaves the inositol head group from PI(4,5)P₂ to produce second messenger, IP3 and DAG (Rhee, 2001). Parallel to this pathway, PI3K phosphorylates PI(4,5)P₂ to generate PI(3,4,5)P₃. Both enzymatic reactions reduce the amplitude and duration of PI(4,5)P₂ spikes. By understanding how lipid kinases, phosphatases, and sequestering molecules indirectly regulate the activity of PIP5K through modulation of membrane lipid composition, we will gain a deeper understanding of how cells control PI(4,5)P₂ lipid homeostasis. Considering the myriad of human diseases linked to the disruption of PIP lipid homeostasis, there is much to gain by understanding the molecular mechanisms that regulate the spatiotemporal dynamics of PIP5K lipid kinase activity in cells.

Materials and methods

Molecular biology

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The gene coding for Legionella pneomophila DrrA/SidM (accession #Q5ZSQ3.1) was used to clone the minimal PI(4)P lipid-binding domain, which is referred to as DrrA throughout the article and Hansen et al., 2019 . Human phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1 PH domain (PLCδ, accession #P51178.2), human oculocerebrorenal syndrome of Lowe inositol polyphosphate 5-phosphatase (OCRL; UniProt #Q01968), human phosphatidylinositol 5-phosphate 4-kinase type-2 beta (PIP4KB; UniProt #P78356), and human phosphatidylinositol 4-phosphate 5-kinase type-1 beta isoform 2 (PIP5KB; UniProt #O14986) were derived from codon-optimized genes synthesized by GeneArt (Invitrogen). The gene encoding yeast Mss4 (UniProt #P38994) was obtained by PCR amplification from yeast genomic DNA (S288C, Saccharomyces cerevisiae). The gene encoding mouse PIP5KA isoform 1 was purchased as a cDNA clone from Horizon Discovery (Cat# MMM1013-202762630, UniProt #P70182). The gene encoding human PIP5KC1 (UniProt #O60331) was provided by Peter Bieling (Max Planck Institute of Molecular Physiology, Dortmund, Germany). The plasmids containing codon-optimized zebrafish PIP5KA (zPIP5KA 49-431aa, UniProt #A0A2R8QMJ9, accession # NP_001018438.1) for expression in bacteria were kindly provided by Jian Hu (Michigan State University, Department of Biochemistry and Molecular Biology). Gene sequences were subcloned into either bacterial, insect cell, or mammalian expression vectors using ligation-independent cloning or Gibson assembly (Gibson et al., 2009). The open-reading frame of all vectors used in this study was sequenced to ensure that protein expression constructs lacked deleterious mutations.

Note that there is some confusion in the literature concerning the naming of mouse and human PIP5K homologs. For example, some articles refer to the human α form (i.e., hPIP5KA) as the mouse β form (i.e., mPIP5KB). This confusion appears to have been corrected in the UniProt gene and protein sequence database, thus making mouse PIP5KA (UniProt #P70182) the direct homolog of human PIP5KA (UniProt #Q99755). Similarly, mouse PIP5KB (UniProt #P70181) the direct homolog of human PIP5KB (UniProt #O14986). In this study, the sequence of mouse PIP5KA used is homologous to human PIP5KA and distinct from human PIP5KB. Refer to Supplementary file 1 for the exact amino acid sequences used for recombinant protein expression and transient transfection in HEK293T cells.

Purification of BACMID DNA

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To create BACMID DNA, FASTBac1 plasmids were transformed into DH10 Bac cells and plated on agar containing 50 µg/mL kanamycin, 10 µg/mL tetracycline, 7 µg/mL gentamycin, 40 µg/mL X-GAL, and 40 µg/mL IPTG. After 2–3 days of growth at 37°C, positive clones were isolated based on blue-white colony selection. Single white colonies were picked and struck on a BACMID agar plate for a second round of selection. BACMIDs were purified from 3 mL bacterial cultures grown overnight in TPM. Bacteria were centrifuged and resuspended in 300 µL of buffer containing 50 mM Tris (pH 8.0), 10 mM EDTA, 100 µg/mL RNase A (QIAGEN PI buffer). Bacteria were then lysed by adding 300 µL of buffer containing 200 mM NaOH, 1% SDS (QIAGEN P2 buffer). Lysis buffer was neutralized by adding 300 µL of 4.2 M guanidine HCl, 0.9 M KOAc (pH 4.8) (QIAGEN N3 buffer). Sample was centrifuged at 23°C for 10 min at 14,000 × g. Supernatant was removed and combined with 700 µL 100% isopropanol. Sample was centrifuged at 23°C for 10 min at 14,000 × g. Supernatant was removed and 200 µL of 70% ethanol was added. Sample was centrifuged at 23°C for 10 min at 14,000 × g. Supernatant was removed and 50 µL of 70% ethanol was added. Sample was centrifuged at 23°C for 10 min at 14,000 × g. Supernatant was removed and DNA pellet was slightly dried in biosafety hood. DNA was solubilized with 40 µL of sterile water. DNA pellet was resuspended by tapping side of micro-centrifuge tube 15–20 times. Concentration of DNA was quantified using NanoDrop (typically 200–300 ng/µL). BACMID DNA was immediately used for transfection of Sf9 cells. This cell line was derived from the Spodoptera frugiperda (Sf9) cell line IPLB-Sf-21-AE (Expression Systems, Cat# 94-001F). The remaining BACMID DNA can be stored in –20°C freezer.

Baculovirus production

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Baculoviruses were generated by transfecting 1 × 10⁶ S. frugiperda (Sf9) insect cells plated for 24 hr in a Corning 6-well plastic dish (Cat# 07-200-80) containing 2 mL of ESF 921 Serum-Free Insect Cell Culture media (Expression Systems, Cat# 96-001, Davis, CA). All media contain 1× concentration of Antibiotic-Antimycotic (Gibco/Invitrogen, Cat# 15240-062). For transfection, 5–7 µg BACMID DNA was incubated with 4 µL Fugene (Thermo Fisher, Cat# 10362100) in 200 µL of ESF 921 media for 30 min at 23°C. BACMID DNA and Fugene were added dropwise to 6-well dish. Media change before and after addition of transfection reagent is unnecessary. After 4–5 days of transfection, viral supernatant (termed ‘P0’) was harvested, centrifuged, and used to infect 7 × 10⁶ Sf9 cells plated for 24 hr in 10 cm tissue culture grade Petri dish containing 10 mL of ESF 921 media and 10% fetal bovine serum (Seradigm, Cat# 1500-500, Lot# 176B14). After 4 days, viral supernatant (termed ‘P1’) was harvested and centrifuged to remove cell debris. Typical P1 viral titer yield is 10–12 mL. The P1 viral titer was expanded in 100 mL Sf9 cell culture grown to a density of 1.25–1.5 × 10⁶ cells/mL in a sterile 250 mL polycarbonate Erlenmeyer flask with vented cap (Corning, #431144). We typically transduced 100 mL Sf9 culture with a concentration of 1% vol/vol of PI viral titer. The remaining PI virus was frozen as 1.5 mL aliquots that were stored in the –80°C freezer. The 10% fetal bovine serum served as a cryo-protectant. After 4 days, viral supernatant (termed ‘P2’) was harvested, centrifuged, and 0.22 µm filtered in 150 mL filter-top bottle (Corning, polyethersulfone [PES], Cat# 431153). The P2 viral supernatant is used for protein expression in High Five cells grown in ESF 921 Serum-Free Insect Cell Culture media. The multiplicity of infection (MOI) for optimal protein expression is determined empirically to minimize cell death and maximize protein yield (typically 1.5–2% vol/vol final concentration of P2 virus).

Protein purification

PIP5KA, PIP5KB, PIP5KC, zPIP5KA, yMss4

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Gene sequences encoding PIP5K family proteins and dimer interface mutants were cloned into FastBac1 vectors in frame with an N-terminal his6-MBP-(Asn)₁₀-TEV-GGGGG. Protein expression in High Five insect cells was constitutive under the polyhedrin (pH) promoter. High Five cells were infected with baculovirus using an optimized MOI, typically 1.5–2% vol/vol, was determined empirically from small-scale test expression (25–50 mL culture). Infected cells were typically grown for 48 hr at 27°C in ESF 921 Serum-Free Insect Cell Culture medium (Expression Systems, Cat# 96-001-01). Cells were harvested by centrifugation, washed with 1× PBS (pH 7.2), and then stored in the –80°C freezer. Flash freezing the cell pellet with liquid nitrogen prior to storage was not essential to purify functional kinase. For purification, frozen insect cell pellets for 4–6 L of liquid culture were thawed in an ambient water bath and lysed into buffer containing 50 mM Na₂HPO₄ (pH 8.0), 10 mM imidazole, 400 mM NaCl, 1 mM PMSF, 5 mM BME, 100 µg/mL DNase, 1 Sigma protease inhibitor cocktail EDTA free per 100 mL lysis buffer using a dounce homogenizer. Lysate was centrifuged at 35,000 rpm (140,000 × g) for 60 min under vacuum using a Beckman Ti-45 rotor at 4°C. Lysate was then batch bound to 5 mL of Ni-NTA Agarose (QIAGEN, Cat# 30230) resin at 4°C for 1–2 hr in a beaker set on a stir plate. Resin was then collected in 50 mL tubes, centrifuged, and washed with buffer containing 50 mM Na₂HPO₄ (pH 8.0), 10 mM imidazole, 400 mM NaCl, and 5 mM BME before being transferred to gravity flow column. NiNTA resin with his6-MBP-(Asn)₁₀-TEV-GGGGG-PIP5K (or yeast Mss4) was then washed with 100 mL of 50 mM Na₂HPO₄ (pH 8.0), 30 mM imidazole, 400 mM NaCl, and 5 mM BME buffer and then eluted into buffer containing 500 mM imidazole. Peak fractions were pooled, combined with 200 µg/mL his6-TEV(S291V) protease, and dialyzed against 4 L of buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 2.5 mM BME for 16–18 hr at 4°C. Dialysate was then combined 1:1 with 20 mM Tris (pH 8.0), 1 mM DTT (~100 mM NaCl final). Precipitation was removed by centrifugation and 0.22 µm syringe filtration. Clarified dialysate was then bound to a MonoS cation-exchange column (GE Healthcare, Cat# 17-5168-01) equilibrated in 20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM TCEP buffer. Proteins were resolved over a 10–100% linear gradient (0.1–1 M NaCl, 45 CV, 45 mL total, 1 mL/min flow rate). PIP5K homologs and paralogs typically eluted from the MonoS column in the presence of 370–450 mM NaCl. Peak fractions containing PIP5K (or Mss4) were pooled, concentrated in a 30 kDa MWCO Vivaspin 6 centrifuge tube (GE Healthcare, Cat# 28-9323-17), and loaded onto a 24 mL Superdex 200 10/300 GL (GE Healthcare, Cat# 17-5174-01) size-exclusion column equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions were concentrated in a 30 kDa MWCO Vivaspin 6 centrifuge tube and snap-frozen at a final concentration of 20–40 µM using liquid nitrogen.

PIP4K2B

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Codon-optimized gene sequence encoding human PIP4K2B isoform 2 (UniProt # P78356) was cloned into a pETM-derived bacterial expression vector to create the following fusion protein: his6-SUMO3-GGGGG-PIP4K2B (1-416aa). Throughout the article, PIP4K2B is referred to as PIP4K. Recombinant PIP4K2B was expressed in BL21(DE3) Star Escherichia coli (i.e., lack endonuclease for increased mRNA stability). Using 2–4 L of Terrific Broth, bacterial cultures were grown at 37°C until OD₆₀₀ = 0.6. Cultures were then shifted to 18°C for 1 hr to cool down. Protein expression was induced with 50 µM IPTG and bacteria-expressed protein for 20 hr at 18°C before being harvested by centrifugation. For purification, cells were lysed into buffer containing 50 mM Na₂HPO₄ (pH 8.0), 400 mM NaCl, 0.4 mM BME, 1 mM PMSF (add twice, 15 min intervals), DNase, 1 mg/mL lysozyme using a microtip sonicator. Lysate was centrifuged at 16,000 rpm (35,172 × g) for 60 min in a Beckman JA-17 rotor chilled to 4°C. Lysate was circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) that had been equilibrated with 100 mM CoCl₂ for 1 hr, washed with Milli-Q water, and followed by buffer containing 50 mM Na₂HPO₄ (pH 8.0), 400 mM NaCl, 0.4 mM BME. Recombinant PIP4K2B was eluted with a linear gradient of imidazole (0–500 mM, 8 CV, 40 mL total, 2 mL/min flow rate). Peak fractions were pooled, combined with 50 µg/mL of his6-SenP2 (SUMO protease), and dialyzed against 4 L of buffer containing 25 mM Na₂HPO₄ (pH 8.0), 400 mM NaCl, and 0.4 mM BME for 16–18 hr at 4°C. Following overnight cleavage of the SUMO3 tag, dialysate-containing his6-SUMO3, his6-SenP2, and GGGGG-PIP4K2B was recirculated for at least 1 hr over a 5 mL HiTrap (Co²⁺) Chelating column. Flow-through containing GGGGG-PIP4K2B was then concentrated in a 30 kDa MWCO Vivaspin 6 before loading onto a Superdex 200 size-exclusion column equilibrated in 20 mM HEPES (pH 7), 200 mM NaCl, 10% glycerol, 1 mM TCEP. In some cases, cation-exchange chromatography was used to increase the purity of GGGGG-PIP4K2B before loading on the Superdex 200. In those cases, we equilibrated a MonoS column 20 mM HEPES (pH 7), 100 mM NaCl, 1 mM TCEP buffer. PIP4K2B (pI = 6.9) bound to the MonoS was resolved over a 10–100% linear gradient (0.1–1 M NaCl, 30 CV, 30 mL total, 1.5 mL/min flow rate). Peak fractions collected from the Superdex 200 were concentrated in a 30 kDa MWCO Vivaspin 6 centrifuge tube and snap-frozen at a final concentration of 20–80 µM using liquid nitrogen.

PLCδ-PH domain

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The coding sequence of human PLCδ-PH domain (11-140aa) was expressed in BL21 (DE3) Star E. coli as a his₆-SUMO3-(Gly)₅-PLCd (11-140aa) fusion protein. Bacteria were grown at 37°C in Terrific Broth to an OD₆₀₀ of 0.8. Cultures were shifted to 18°C for 1 hr, induced with 0.1 mM IPTG, and allowed to express protein for 20 hr at 18°C before being harvested. Cells were lysed into 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, 0.4 mM BME, 1 mM PMSF, 100 µg/mL DNase using a microfluidizer. Lysate was then centrifuged at 16,000 rpm (35,172 × g) for 60 min in a Beckman JA-17 rotor chilled to 4°C. Lysate was circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) charged with 100 mM CoCl₂ for 1 hr. Bound protein was then eluted with a linear gradient of imidazole (0–500 mM, 8 CV, 40 mL total, 2 mL/min flow rate). Peak fractions were pooled, combined with SUMO protease (50 µg/mL final concentration), and dialyzed against 4 L of buffer containing 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, and 0.4 mM BME for 16–18 hr at 4°C. Dialysate-containing SUMO cleaved protein was recirculated for 1 hr over a 5 mL HiTrap Chelating column. Flow-through containing (Gly)₅-PLCδ (11-140aa) was then concentrated in a 5 kDa MWCO Vivaspin 20 before being loaded on a Superdex 75 size-exclusion column equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions containing (Gly)₅-PLCδ (11-140aa) were pooled and concentrated to a maximum concentration of 75 µM (1.2 mg/mL) before snap-freezing with liquid nitrogen and storage at –80°C.

OCRL and DrrA

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The coding sequence of human 5-phosphatase OCRL (234-539aa of 901aa isoform) was expressed in BL21 (DE3) E. coli as a his6-MBP-(Asn)₁₀-TEV-(Gly)₅-OCRL fusion protein. DrrA/SidM (544-647aa of 647aa gene) derived from L. pneomophila was expressed in BL21 (DE3) E. coli as a his6-MBP-(Asn)₁₀-TEV-(Gly)₅-DrrA(544-647aa) fusion protein. For the proteins described above, bacteria were grown at 37°C in Terrific Broth to an OD₆₀₀ of 0.8. Cultures were shifted to 18°C for 1 hr, induced with 0.1 mM IPTG, and allowed to express protein for 20 hr at 18°C before being harvested. Cells were lysed into 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, 0.4 mM BME, 1 mM PMSF, 100 µg/mL DNase using a microfluidizer. Lysate was then centrifuged at 16,000 rpm (35,172 × g) for 60 min in a Beckman JA-17 rotor chilled to 4°C. Lysate was circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) charged with 100 mM CoCl₂ for 1 hr. Bound protein was then eluted with a linear gradient of imidazole (0–500 mM, 8 CV, 40 mL total, 2 mL/min flow rate). Peak fractions were pooled, combined with TEV protease (75 µg/mL final concentration), and dialyzed against 4 L of buffer containing 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, and 0.4 mM BME for 16–18 hr at 4°C. Dialysate-containing TEV protease cleaved protein was recirculated for 1 hr over a 5 mL HiTrap Chelating column. Flow-through containing (Gly)₅-protein was then concentrated in a 5 kDa MWCO Vivaspin 20 before being loaded on a Superdex 75 (10/300 GL) size-exclusion column equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions were pooled and concentrated before snap-freezing in liquid nitrogen.

DrrA-OCRL

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Chimeric 5-phosphatase his6-MBP-(Asn)₁₀-TEV-(Gly)₅-DrrA(544-647aa)-(Gly)₅-OCRL was expressed in BL21 (DE3) E. coli. For the proteins described above, bacteria were grown at 37°C in Terrific Broth to an OD₆₀₀ of 0.8. Cultures were shifted to 18°C for 1 hr, induced with 0.1 mM IPTG, and allowed to express protein for 20 hr at 18°C before being harvested. Cells were lysed into 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, 0.4 mM BME, 1 mM PMSF, 100 µg/mL DNase using a microfluidizer. Lysate was then centrifuged at 16,000 rpm (35,172 × g) for 60 min in a Beckman JA-17 rotor chilled to 4°C. Lysate was circulated over 5 mL HiTrap Chelating column charged with 100 mM CoCl₂ for 1 hr. Bound protein was then eluted with a linear gradient of imidazole (0–500 mM, 8 CV, 40 mL total, 2 mL/min flow rate). Peak fractions were pooled, combined with TEV protease (75 µg/mL final concentration), and dialyzed against 4 L of buffer containing 50 mM Na₂HPO₄ (pH 8.0), 300 mM NaCl, and 0.4 mM BME for 16–18 hr at 4°C. Dialysate-containing TEV protease cleaved protein was recirculated for 1 hr over a 5 mL HiTrap Chelating column. Flow-through containing (Gly)₅-DrrA(544-647aa)-(Gly)₅-OCRL were then buffer exchanged into 20 mM HEPES pH 7, 100 mM NaCl, 1 mM DTT using a HiPrep 26/10 desalting column (GE Healthcare, Cat# 17-5087-01). DrrA-OCRL was then loaded onto a 1 mL MonoS (5/50 GL) cation-exchange column (GE Healthcare, Cat# 17-5168-01) equilibrated in 20 mM HEPES pH 7, 100 mM NaCl, 1 mM DTT. DrrA-OCRL was separated from impurities by applying a linear salt gradient (0.1–1 M NaCl) over 45 CV (45 mL total). DrrA-OCRL eluted from the MonoS column in the presence of 250–300 mM NaCl. Peak fractions were pooled and concentrated in a 10 kDa MWCO Vivaspin 6 before being loaded on a Superdex 75 (10/300 GL) size-exclusion column equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions were pooled and concentrated before snap-freezing in liquid nitrogen.

Sortase-mediated peptide ligation

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All lipid sensors and catalytic domains were labeled on a N-terminal (Gly)₅ motif using Sortase-mediated peptide ligation (Ton-That et al., 1999; Guimaraes et al., 2013). We devised a novel approach for chemically modifying an LPETGG peptide with fluorescent dyes, which we then conjugated to our protein of interest. The LPETGG peptide was synthesized to >95% purity by ELIM Biopharmaceutical (Hayward, CA) and labeled on the N-terminal amine with N-hydroxysuccinimide (NHS) fluorescent dye derivatives (e.g., NHS-Alexa488). This was achieved by combining 10 mM LPETGG peptide, 15 mM NHS-Alexa488 (or other fluorescent derivatives), and 30 mM triethylamine (Sigma, Cat# 471283) in anhydrous DMSO (Sigma, Cat# 276855). This reaction was incubated overnight in the dark at 23°C before being stored in a –20°C freezer. Prior to labeling (Gly)₅-containing proteins, unreacted NHS-Alexa488 remaining in the LPETGG labeling reaction was quenched with 50 mM tris(hydroxymethyl)aminomethane (Tris) (pH 8.0) buffer for at least 6 hr. Complete quenching of unreacted NHS-Alexa488 was verified by the inability to label (Gly)₅-containing proteins in the absence of a Sortase.

When labeling (Gly)₅-containing proteins with the fluorescently labeled LPETGG peptide, we typically combined the following reagents: 50 mM Tris (pH 8.0), 150 mM NaCl, 50 µM (Gly)₅-protein, 500 µM Alexa488-LPETGG, and 10–15 µM His₆-Sortase (Δ57; lacks first 57 amino acids). This reaction mixture was incubated at 16–18°C for 16–20 hr, before buffer exchange with a G25 Sephadex column (e.g., PD10 or NAP5) to remove majority of dye and dye peptide. The his₆-Sortase was then captured on NiNTA agarose resin (QIAGEN) and unbound, labeled protein was separated from remaining fluorescent dye and peptide using a Superdex 75 or Superdex 200 size-exclusion column (24 mL bed volume).

Preparation of small unilamellar vesicles

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The following lipids were used to generate small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P₂, Avanti # 840046X), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (16:0 Liss Rhod PE, Avanti # 810158C), and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C). In the main text, 16:0 Liss Rhod PE is referred to as Rhod PE. Lipids were purchased as single-use ampules containing between 0.1 and 5 mg of lipids dissolved in chloroform. Brain PI(4)P and PI(4,5)P₂ were purchased as 0.25 mg/mL stocks dissolved in chloroform:methanol:water (20:9:1). To make liposomes, 2 µmole total lipids were combined in a 35 mL glass round-bottom flask containing 2 mL of chloroform. Lipids were dried to a thin film using rotary evaporation with the glass round-bottom flask submerged in a 42°C water bath. After evaporating all the chloroform, the round-bottom flask was flushed with nitrogen gas for at least 30 min. Lipid film was resuspended in 2 mL of PBS (pH 7.2), making a final concentration of 1 mM total lipids. All lipid mixtures expressed as percentages (e.g., 98% DOPC, 2% PI(4)P) are equivalent to molar fractions. For example, a 1 mM lipid mixture containing 98% DOPC and 2% PI(4)P is equivalent to 0.98 mM DOPC and 0.02 mM PI(4)P. To generate 30–50 nm SUVs, 1 mM total lipid mixtures were extruded through a 0.03 µm pore size 19 mm polycarbonate membrane (Avanti #610002) with filter supports (Avanti #610014) on both sides of the PC membrane. Hydrated lipids at a concentration of 1 mM were extruded through the PC membrane 11 times.

Preparation of supported lipid bilayers

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SLBs are formed on 25 × 75 mm coverglass (IBIDI, #10812). Coverglass is first cleaned with 2% Hellmanex III (Fisher, Cat# 14-385-864) and heated to 60–70°C in a glass coplin jar. It is then incubated for at least 30 min. Coverglass is then washed extensively with Milli-Q water and then etched with Piranha solution (1:3, hydrogen peroxide:sulfuric acid) for 10–15 min the same day and SLBs were formed. Etched coverglass, in water, is rapidly dried with nitrogen gas before adhering to a 6-well sticky-side chamber (IBIDI, Cat# 80608). SLBs are formed by flowing 30 nm SUVs diluted in PBS (pH 7.2) to a total lipid concentration of 0.25 mM. After 30 min, IBIDI chambers are washed with 5 mL of PBS (pH 7.2) to remove non-absorbed SUVs. Membrane defects are blocked for 15 min with a 1 mg/mL beta-casein (Thermo Fisher Scientific, Cat# 37528) diluted in 1× PBS (pH 7.4). Before use as a blocking protein, frozen 10 mg/mL beta-casein stocks were thawed, centrifuged for 30 min at 21,370 × g, and 0.22 µm syringe filtered. After blocking SLBs with beta-casein, membranes were washed again with 1 mL of PBS, followed by 1 mL of kinase buffer before TIRF-M.

Kinetics measurements of PIP lipid phosphorylation

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The kinetics of PI(4)P phosphorylation was measured on SLBs formed in IBIDI chambers and visualized using TIRF microscopy. Reaction buffer contained 20 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM ATP, 5 mM MgCl₂, 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta-casein (Thermo Scientific, Cat# 37528), 20 mM BME, 320 µg/mL glucose oxidase (Serva, #22780.01 Aspergillus niger), 50 µg/mL catalase (Sigma, #C40-100MG Bovine Liver), and 2 mM Trolox (UV treated, see methods below). Perishable reagents (i.e., glucose oxidase, catalase, and Trolox) were added 5–10 min before image acquisition. For all experiments, we monitored the change in PI(4)P or PI(4,5)P₂ membrane density using a solution concentration of 20 nM Alexa647-DrrA(544-647) or 20 nM Alexa488-PLCδ, respectively. We calculated the density of PIP lipids (lipids/µm²) assuming a footprint of 0.72 nm² for DOPC lipids (Galush et al., 2008; Vacklin et al., 2005).

Single-molecule cell lysate assay

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Genes encoding mPIP5KA (5KA), hPIP5KB (5KA), hPIP5KC (5KC), zPIP5KA (z5KA), yMss4, and hPIP4K2B (4KB) were cloned into lentiviral expression vectors containing an SFFV promoter to drive expression of N-terminal mNG fusion proteins in mammalian cells. HEK293T cells were transfected with 15 µg of plasmid DNA encoding mNG-tagged lipid kinases, plus 30 µg polyethylenimine (PEI) diluted into 0.5 mL Opti-MEM. Prior to transfection, HEK293T cells were grown to a confluency of 50–60% in 10 cm dishes in DMEM GlutaMAX media (Thermo Fisher, Cat #10566016) containing 10% FBS and penicillin/streptomycin. After 20–24 hr for transfection, adherent HEK293T grown in 10 cm dishes were washed with 5 mL 1× PBS (pH 7.4). After vacuum aspiration of the PBS, cells were incubated in 1 mL of CellStripper (Corning, Cat# 25-056Cl) for 10 min at room temperature. Detached cells were resuspended in 9 mL of PBS and transferred to a 15 mL conical tube. Cells were centrifuged for 5 min at 500 rcf at 4°C. The cell pellet was resuspended in 1 mL of PBS, transferred to a microcentrifuge tube, and centrifuged for 3 min at 500 rcf. After removing PBS by vacuum aspiration, cell pellets were resuspended in 0.6 mL of lysis buffer containing 20 mM HEPES pH 7, 150 mM NaCl, 5 mM MgCl₂, Sigma protease inhibitor (Cat# P3840, 1% vol/vol final), Sigma phosphatase inhibitor 2 (0.5% vol/vol final), Sigma PPtase inhibitor 3 (0.5% vol/vol final), 50 mM NaF, 15 µg/mL benzamidine, and 1 mM PMSF. Microtip sonication was used to rupture transfected cell on ice using the following program: 20% amplitude, 2 s ON and 20 s OFF for 15 cycles. Cell lysate was centrifuged for 45 min at 21,300 rcf at 4°C. Following centrifugation, 75% of the supernatant was transferred to new 1.7 mL microcentrifuge tube. The clarified lysate was then mixed 4:1 with lysis buffer containing 50% glycerol (vol/vol). This resulted in lysate containing a final glycerol concentration of 10% (vol/vol). Cell lysate was aliquoted and flash-frozen in liquid nitrogen. We did not observe any difference in the quality of the mNG-labeled protein comparing fresh versus freeze-thawed cell lysate.

To determine the concentration of mNG-tagged lipid kinase in HEK293T cell lysate we generated a twofold serial dilution of bacterially purified mNG diluted in 1× PBS (pH 7.4) and 0.1% NP-40 detergent. The fluorescence intensity was measured using a BioTek 96-well format using a plate reader to generate a standard curve for fluorescence intensity as a function of mNG concentration (Figure 1—figure supplement 2). mNG was excited with a 500 nm light using a 500/10 nm bandpass filter. The emission was monitored at 517 nm with high photomultiplier tube (PMT) sensitivity.

Quantitative fluorescence microscopy using supported lipid bilayer standards

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We measured the membrane surface density of Ax647-PIP5KB (Figure 2) using previously described methods (Galush et al., 2008). In brief, we titrated the molar fraction of Atto655-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Atto655-DPPE; Atto-TEC, Cat# AD 655-151) against DOPC lipids. SUVs were formed using microtip sonication. We used TIRF microscopy to measure the fluorescence intensity of SLBs containing varying concentrations of Atto655-DPPE. These values were used to generate a standard curve that was used to calculate the surface density of membrane-bound Alexa647-PIP5KB. In order to compare the fluorescence intensity of Alexa647-PIP5KB to Atto655-DPPE, we calculated the scaling factor that accounts for the difference in molecular brightness of the two fluorophores measured on the same microscope with identical camera settings, laser power, filters, and optics. To determine the scaling factor, we measured the fluorescence intensity of solutions containing identical concentrations of either Alexa647-PIP5KB or SUVs containing the same molar concentration of Atto655-DPPE (Figure 2—figure supplement 3). These solutions were added to an imaging chamber passivation with a supported membrane (95% DOPC and 5% DOPS) in order to prevent nonspecific absorption of Alexa647-PIP5KB and SUVs containing Atto655-DPPE. To measure the solution intensity of the Alexa647-PIP5KB and Atto655-DPPE-containing samples, we choose a z-axis imaging plane that was 5 µm above the glass surface and acquired at least 20 fluorescence measurements for each solution concentration. These fluorescence intensity values were averaged and plotted as a function of the fluorophore solution concentration. The scaling factor was calculated by dividing the slope of Alexa647-PIP5KB plot by the slope of the Atto655-DPPE plot.

Surface density calibration of mNG-PIP5KB

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Densities of mNG-PIP5KB were estimated using a surface density calibration curve of mNG attached to the lipid bilayer. SLB containing 96% DOPC and 4% Ni-NTA-DOGS by molar percent was incubated with His6-mNG with concentrations ranging from 0.1 nM to 10 nM in 20 mM HEPES (pH 7.0), 150 mM NaCl, 200 µg/mL beta-casein, 5 mM BME for 30 min. The chambers were rinsed with 1 mL of buffer and a calibration curve was established between the TIRF average intensity and surface densities measured by FCS of mNG on the membrane using previously described methods (Chung et al., 2019; Chung et al., 2018). The same calibration curve was used to estimate the surface density of mNG-PIP5KB. The kinetic measurements were performed on SLB containing 96% DOPC and 4% PI(4)P by molar percent, with the presence of 20 nM Cy3-PLCδ to monitor the change of PI(4,5)P₂. The total PIP lipid was calculated to be at 55,555 lipids/µm². The start and end of the reaction were approximated to have 0 and 55,555 lipids/µm² of PI(4,5)P₂, respectively. Images were acquired at a 2 s interval. PI(4,5)P₂ and mNG-PIP5KB surface density was calculated for each time point. The $\frac{d P I (4,5) P 2}{d t}$ for each time point was obtained by using the slope of linear regression of PI(4,5)P₂ level change in a +2 s to –2 s time range.

Feedback analysis of PIP5K

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Alexa488-PLCδ intensity was measured from TIRF images acquired at a 2 s interval. The reaction coordinate (x) for each time point was calculated by normalizing the start intensity to 0 and end intensity to 1. The $\frac{d x}{d t}$ for each time point was obtained by using the slope of linear regression of reaction coordinate change in a +2 s to –2 s time range.

Chromium-patterned glass coverslips

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25 × 75 mm No. 1.5 thickness glass coverslips were cleaned in acetone by sonication, then washed with Milli-Q water extensively. The coverslips were dried by nitrogen gas, then baked on 120°C hot plate for 5 min. S1805-positive photoresist were spin-coated on the coverslips by spinning for 2 s at 500 rpm (ACL 440), then for 30 s at 4111 rpm (ACL 3900). The photoresist on the edge of the coverslips were removed by cotton swap soaked with acetone, then baked on 120°C hot plate for 1 min. Mask with desired pattern was mounted on an OAI Series 200 Aligner. The photoresist-coated coverslip was exposed for 0.6 s with UV power around 30 mJ, then developed with MicroPosit MF-321 Liquid Developer for 40 s with mild shaking. The developed coverslips were rinsed with water and dried with nitrogen gas. Approximately 9-nM-thick chromium was subsequently deposited on the coverslips using an electron beam evaporator at 1 × 10^–6 torr. Finally, photoresist is lifted from chromium-patterned glass substrates by bath sonication in Dow Electronic Materials MicroPosit Remover 1165 for 10 min two times, then washed with water.

Microscope hardware and imaging acquisition

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Single-molecule imaging experiments were performed on an inverted Nikon Eclipse Ti and Ti2 microscopes using a ×100 Nikon objective (1.49 NA) oil immersion TIRF objective. The macroscopic spatial patterning of PIP compositional patterns and the chromium-patterned SLB were visualized using a ×60 Apo TIRF oil immersion objective (1.45 NA). The x-axis and y-axis positions were manually controlled using an ASI stage and joystick. All images were acquired using either a iXon Ultra or iXion Life 897 EMCCD camera (Andor Technology Ltd, UK). Fluorescently labeled proteins were excited with either a 488 nm, 561 nm, or 637 nm diode laser (OBIS laser diode, Coherent Inc, Santa Clara, CA) controlled with either a Solemere (Nikon Ti) or Vortran (Nikon Ti2) laser drive with acousto-optic tunable filter (AOTF) control. The power output measured through the objective for single-particle imaging was 1–3 mW. For dual-color imaging of spatial PIP lipid patterns on SLBs, samples were excited with 0.2–0.5 mW 488 nm and 0.2–0.5 mW 637 nm light, as measured through the objective. Excitation light was passed through the following dichroic filter cubes before illuminating the sample: (1) ZT488/647rpc and (2) ZT561rdc (ET575LP) (Semrock). Fluorescence emission was detected on an ANDOR EMCCD camera position after a Sutter emission filter wheel housing the following emission filters: ET525/50M, ET600/50M, ET700/75M (Semrock). All experiments were performed at room temperature (23°C). Microscope hardware was controlled using both Micro-Manager v4.0 (Edelstein et al., 2010) and Nikon NIS elements.

Single-particle tracking

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Fluorescent particle detection and tracking was performed using the ImageJ/Fiji TrackMate plugin (Jaqaman et al., 2008). Image stacks containing ~1000 16-bit image stacks in the form of a .nd2 file or .tif stack were loaded in ImageJ. Image sequences were cropped to 400 × 400 pixels in order to minimize differences in field illumination caused by TIRF illumination. Using the LoG detector option, particles were identified based on brightness and their signal-to-noise ratio. After identifying the position of all fluorescent particles, we used the LAP tracker to generate particle trajectories that followed molecular displacement as a function of time. Particle trajectories were then filtered based on Track Start (removed trajectories that began in first frame), Track End (removed trajectories present in last frame), Duration (removed trajectories ≤2 frames), Track displacement (removed immobilized particles), and X – Y location (removed particles near the edge of the images). Trajectories were filtered to remove 1–5% of particles that were immobilized throughout the image sequence. The TrackMate output files were analyzed using Prism to calculate the single-molecule dwell times and diffusion coefficients.

Step-size distribution of single-particle trajectories was plotted in Prism. For all analyses presented in this article, the bin size for the step-size distribution equals 0.01 µm. For curving fitting, the step-size distributions were plotted as probability density versus step size (µm). This was achieved by dividing the frequency distribution (i.e., y-axis values) by the bin size (0.01 µm). The probability density versus step-size plots were fit to the following one- or two-species distributions:

Single-species model:

f (r) = \frac{r}{2 D τ} e^{- (\frac{r^{2}}{4 D τ})}

Two-species model:

f (r) = α \frac{r}{2 D_{1} τ} e^{- (\frac{r^{2}}{4 D_{1} τ})} + (1 - α) \frac{r}{2 D_{2} τ} e^{- (\frac{r^{2}}{4 D_{2} τ})}

Variables are defined as the D1 = diffusion coefficient species 1 (µm²/s), D2 = diffusion coefficient species 2 (µm²/s), alpha (τ₁ = % of species 1, r = step size (µm)), τ₂ = time interval between steps (s). Final step-size distribution plots were generated in Prism graphing software and using the following equations: (1 species model): f(x) = x/(2*D1*t)*exp(-(x^2/ (4*D1*t))), (2 species model): f(x) = alpha*(x/(2*D1*t)*exp(-(x^2/ (4*D1*t)))) + (1-alpha)*(x/(2*D2*t)*exp(-(x^2/ (4*D2*t)))).

During our characterization of membrane-bound mNG-PIP5K and Ax647-PIP5K, we established conditions that caused fluorescently labeled PIP5K to diffuse more slowly. This includes the use of expired or oxidize PI(4,5)P₂ lipids, as well as membrane blocking with 1 mg/mL beta-casein for >15 min. For both sets of conditions, we measured diffusion coefficients for mNG-PIP5K and Ax647-PIP5K that were 0.09–0.12 µm²/s. For the data presented in this article, we controlled for these factors and measured faster diffusion coefficients in the range of 0.14–0.19 µm²/s.

To calculate the single-molecule dwell times for Ax647-PIP4K and Ax647-PIP5K, we generated a cumulative distribution frequency (CDF) plot using the frame interval as the bin size (e.g., 50 ms). The log₁₀(1-CDF) was plotted against the dwell time and fit to either a single- or double-exponential decay curve.

Single-exponential model:

f (t) = e^{(- x / τ)}

Two-exponential model:

f (t) = {α * e}^{(- x / τ 1)} {+ (1 - α) * e}^{(- x / τ 2)}

Fitting procedure was initiated with a single exponential. In case of a low-quality single-exponential fit, a maximum of two-species model was used. For double-exponential fit, alpha (α) represents the fraction of fast-dissociating molecules characterized by τ₁.

Quantification of mNeonGreen fluorescence brightness

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Supported membranes containing 4% PI(4,5)P₂ and 96% DOPC lipids were used to recruit mNG-tagged PIP4K and PIP5K proteins from solution to bilayers. Proteins were excited and visualized by TIRF microscopy using ~1 mW of 488 nm laser power measured through a ×100 Nikon objective (1.49 NA) oil immersion TIRF objective. To determine the molecular brightness distribution, images were acquired from N = 10–30 fields of views. Membrane-bound mNG-tagged kinases were not exposed to 488 nm light until acquiring a single snapshot. This ensured that a negligible amount of photobleaching occurred prior to imaging. Unless stated otherwise, quantification of molecular brightness corresponds to molecules that had not been continuously imaged.

Image analysis, curve fitting, and statistics

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Image analysis was performed using ImageJ. Curve fitting was performed using Prism 9 (GraphPad). Single-molecule dwell time, step size, and molecular brightness distributions presented in this article represent combined data from three technical replicates with 2–3 movies acquired from multiple fields of view for each experimental condition. Dwell time distributions and curve fits were generated with N ≥ 1000 particle trajectories (Figure 2B, Figure 3C–E, Figure 2—figure supplement 2). Step-size distribution plots and curve fits represent ≥10,000 measured displacements (Figure 1J, Figure 2C, Figure 3F, Figure 3—figure supplement 1). Single-molecule brightness distribution plots were generated from analyzing N ≥ 1000 fluorescent particles (Figure 1F–I, Figure 4C, D, G, and H, Figure 1—figure supplement 3, Figure 1—figure supplement 4D and E, Figure 4—figure supplement 1). See Tables 1 and 2, and figure legends for more exact statistics associated with the data presented.

Supplementary file 1

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for all figures.

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Decision letter

Janice L Robertson

Reviewing Editor; Washington University in St Louis, United States
Jonathan A Cooper

Senior Editor; Fred Hutchinson Cancer Research Center, United States
Janice L Robertson

Reviewer; Washington University in St Louis, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Membrane-mediated dimerization potentiates PIP5K lipid kinase activity" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Janice L Robertson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Philip Cole as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

In general, the reviewers found this paper to be of high interest, presenting exciting results leading to a mechanistic understanding of regulation of pattern forming behavior by PIP5K kinase in membranes. While the data presented was mostly convincing, there are concerns about the interpretation of whether this is dimer specific or related to higher-order oligomerization. In addition, the study suffers from a lack of clarity in the writing, as well as inconsistencies and shortcomings in data presentation. Specifically, the following revisions are required:

1) While there is evidence of linkage to oligomerization, the overall interpretation that the mechanism is specifically linked dimerization is not strongly supported. To address this, the studies that are currently presented require additional clarification, and control experiments are necessary to map out the dimer specific signal on membranes. Alternatively, the mechanism may be modified to consider that this effect may come from an ensemble of higher-order oligomeric states. With this, the paper and the title require significant revisions to clarify this point.

2) Sample sizes, errors and statistical tests must be calculated and clearly presented in the paper and in the figure legends. In its current form, it is not possible to assess whether the results presented are reproducible.

3) The writing needs significant revisions to improve coherency and clarity. The manuscript should be revised to improve accessibility for a broad group of readers, with improved background and discussion of the physical concepts and equations that are presented. In addition, the discussion should be revised to better related the current findings to recently published studies.

4) Address the specific comments and questions raised by the reviewers below.

Overall, the study would benefit from editing to improve coherency and clarity.

Reviewer #1 (Recommendations for the authors):

1. The results presented in the size exclusion chromatography analysis (Figure 1B) need some clarification. There is a hypothesized dimer peak for PIP4K, monomer peak for zPIP5KA/D84R and a sharp monodispersed peak for zPIP5KA, that is interpreted as a monomer/dimer mix. While rapid monomer-dimer exchange might account for this, it is surprising that such as mixture is not smeared over both monomer and dimer elution volumes. Changes in column behavior can also create the appearance of peak shifts and so additional calibration is necessary in order to be able to interpret this data. The protein samples should be run with a calibration sample that can control for changes in column behavior and also allow the identification of expected peak positions. In addition, the protein concentration prior to loading should be controlled, as this can also lead to observation of different oligomeric species. An alternate method that often presents clearer results is cross-linking the dilute protein sample by something like glutaraldehyde and the running on SDS-PAGE. This may be used to rule out higher-order oligomeric species.

2. The brightness analysis presented in Figure 1D,E indicates either that the mNG is folded and fluorescent with 100% yield, or that the PIP4KB being observed is a higher order oligomer than dimer. The PIP4KB intensity distribution in Figure 1E shows very little overlap with the monomeric distributions, and a broad intensity distribution that is 2-3 fold greater that the monomeric samples. Considering binomial labeling probabilities, with around 80% labeling efficiency, a dimer is expected to yield nearly equal probabilities of single and double labelled species, which does appear to be the case for the fluorophore labelled protein presented in Figure 1 —figure supplement 2. For the mNG samples, it is rare for the fluorophore to fold with 100% efficiency, and so it is more probable that the resultant intensity distribution corresponds to > 2 proteins. Photobleaching analysis of the spots would reveal clearer information and test whether higher-order oligomeric species are present.

3. The statement of weak monomer-dimer equilibrium is not strongly supported by the presented data. To say this, one would need to show a reversible monomer to dimer conversion as a function of protein density, such as an association isotherm. While the data presented shows clear evidence of monomers, the change in behavior with protein concentration is in line with higher order oligomerization. This is even diagrammed in Figure 2A. To isolate the dimer-specific species signal, functional cross-linking of the protein might be useful to switch the protein from monomeric to dimeric states and characterize the dimer-specific signal at low protein densities.

4. The major argument for the effects being dimerization dependent are from the results of the D51R mutation, as it is interpreted that this mutation solely affects dimerization. However, it seems plausible that other things may change in the protein that impact catalytic activity that are outside of the dimerization. It is unclear as to whether this particular mutant is well folded on its own, thus further analysis of the protein would be useful here. Is the binding and catalytic activity preserved for D51R in solution, and how does this compare to wild-type under monomeric conditions? If dimerization was observable, for example via AUC, then this would also provide supportive evidence of the fold, as well as report the change in stability.

5. The binding curves for PIP5KA to the membrane in Figure 2F are very clear and present interesting results on the different binding capacities as a function of PIP2. Since the lower leg of the monomer-oligomer equilibrium reaction on the membrane (Figure 4D) will be dependent on the membrane associated protein density, it is important to have this full binding relationship for all constructs in order to interpret the results from the different experiments. As such, this information seems essential for the interpretation of the D51R data. While the dwell time and diffusion distributions show similar behavior to WT, this only reports on the behavior of the molecules that are bound and are all likely to be monomers under the examined conditions (Figure 3C,D). It does not directly report on whether there is the same amount of D51R vs. WT on the membrane. In this case, the data presented in Figure 5, is not expected to have the same amount of membrane bound PIP5K, which could account for the differences in behavior rather than dimerization. Indeed, the addition of 1 μm of D51R increases the observation of the compositional patterns.

Reviewer #2 (Recommendations for the authors):

1. It is not a weakness but rather a limitation that the study is not extended experimentally into real cell situations and only speculates about the potential impact of its finding to the control of the enzymes in real cells.

2. Some of the methods and terms should be defined and explained for the general reader: e.g. It would be useful to define "dwell time" or explain what the Y axis is in Figure 2B. (for example what is CDF?).

3. Similarly, the detailed description of how the catalytic activity of the enzymes were calculated (lines 270-310) is important for the experts. However, it would be desirable to "translate" the following sentences to the general reader.

"When the derivative of the PIP5K kinase reaction traces were plotted against the reaction coordinate, x, the curve displayed a high degree of asymmetry that required a second-order term to fit (kWT(x) = k0 + k1x + k2x2) (Figure 4E). In contrast, the dx⁄dt curve for the dimer mutant was parabolic and could be fit using an equation that describes an enzyme with first order positive feedback (kD51R(x) = k0 + k1x) based on product binding (Figure 4E)." This may not be necessary if the paper is published in a biophysical journal, but for most of the readers of eLife, this description is not very accessible.

4. The legend to Figure 4 Supplement 1. does not really describe what is measured here. First of all, what is measured as "auto-correlation" should be explained. It is not clear what the different colors mean (the concentrations refer to what component?). Panel A-C appear to show graphs on the various PIP5KB forms (wt or mutants), but the Figure legend for panel A does not refer to PIP5KB.

5. The same is true for the legend to Figure 4 Supplement 2. From the description, one would expect to see three similar measurements for the three forms of the enzyme. Clearly, panel A shows a curve that is not explained. Panels B and C show FCS density as a function of TIRF intensity. What is measured here (what is FCS density?)? How do these curves inform about lipid kinase activity? Clearly, the Authors should do better to explain what is shown here and how does that translate to kinase activity.

6. I think the DrrA designation of what is better known as SidM should at least be clearly stated somewhere (preferably also in the Figure legends).

7. Remove the prime from all "5'-phosphatase". There are no prime numbers on the inositol ring just simple numbers designating the positions on the single carbon ring.

Reviewer #3 (Recommendations for the authors):

1. Line 150 and Figure 1B: The authors are referring to a dimer mutant of PIP5K. They need to clarify if that means that the protein is locked in its dimerized state or if the dimerization interface is disrupted and thus the protein resides in its monomeric state. If, as indicated by the 'dimer mutant' reference the protein should be locked in a dimer state, why does the SEC elution profile and their annotation rather indicate that the protein elutes as a monomer? Also, it would be beneficial for the reader to indicate the name of the dimer mutant in the text.

2. Figure 1 Supplement 2: Monomer/dimer distribution of mNG-PIP4K in Figure 1E are considerably different to the ones shown for the Alexa488-PIP4K version in supplement 2. Is there a reason for this comparison, especially as the authors are later introducing another chemical label with the Alexa647-conjugation construct? They should rather repeat the experiment with the Alexa647-conjugation construct that they are using later on.

3. Figure 1B: In Figure 1B, the 'dimer' mutant of PIP5K is annotated as zPIP5KA(D84R), however in the respective figure caption, it is referred to as zPIP5KA(D51R) mutant – which one now is the correct mutant? The authors should use a size standard to calibrate the SEC elution profiles to ensure the correct detection of monomers and dimers (SEC-MALS would be even better suited). The critical reader would furthermore like to see an SDS-PAGE gel of the purified proteins, as especially the elution profile of PIP5K indicates impurities.

4. Figure 1C/D: The authors should indicate the abbreviations of both proteins in the respective figure captions.

5. How do the authors account for bleaching in their TIRF single particle tracking assay?

6. Supplementary Figure 1A: What is the reproducibility of the mNG-calibration curve for the quantitative analysis of kinase presence in the cell lysate? As indicated in the figure caption, only a representative curve is shown, however adding standard errors for each concentration and the indication of the number of replicates would need to be included.

7. Lines 180-181: Several mNG-fusion constructs are named, but it is completely unclear for the reader what they are, as they have not been introduced (e.g. mNG-PIP5KA/B/C or mNG-yMss4?). Even more importantly, in Figure 1E, which is the figure that has been referenced in the text, there are again other abbreviations for the used constructs that do not fit the ones named in the text or have not been referenced in the text. Likewise, also no consistency between the abbreviations between Figures 1E and F. the authors need to clarify this and keep the names of constructs consistent throughout the whole text, figures, and supplementary information.

8. Figure 1F: The authors are referring to a step size distribution in the figure caption, but at the same time state diffusion coefficients for the respective constructs – they need to elaborate on how exactly they got the diffusion coefficients from the step size distribution, more specifically: what kind of fitting they used. As in Figure 2C, the fit should also be included in the plot. Furthermore, what is the error and standard deviation for the diffusion coefficients? It would be beneficial for the reader to also state the deviations in lines 190/191.

9. As stated in line 192, the authors are comparing the diffusion coefficients of the cell lysate kinases with purified kinases in Figure 2C. However, in the two cases different membrane compositions have been used – in Figure 1F they state that 2 % PIP2 have been used, while in Figure 2C 4 % have been incorporated. Why do they change the proportion of PIP2 in the membrane between the two cases? This inconsistency especially impedes their assumption in line 193, that the membrane binding of kinases in the cell lysate is indistinguishable from the purified case.

10. Figure 2B: For readers that are not familiar with single particle tracking, the cumulative density function should be introduced in the caption of the figure or even in the axis label. Same comment for Figure 3CDE.

11. Figure 2C: Again, a new abbreviation is being introduced for PIP5KB.

12. Figure 2F: Error bars are missing and SEM of Kd values. In the respective caption, they should state the number of independent replicates. They also need to indicate in the caption that the nh – values they are referring to in the figure are the Hill coefficients. The manuscript would benefit considerably if they would repeat the experiments shown in this Figure also with the D51R mutant – especially as they are comparing the mutant with the wild type throughout the manuscript, and only for Kd values the respective differences cannot be judged. Similar comment for Figure 4AB regarding error bars and replicate numbers.

13. Figure 3CD: The authors need to indicate in the caption or the figure that this is a low protein density condition.

14. Figure 3F: Please state the respective diffusion coefficients you have obtained from the used fit. Same comment for Supplementary Figure 3 (and adjust x-axis label to be consistent with similar graphs).

15. Supplementary Figure 4 (Figure supplement 1 and 2): It seems like the Figure captions of Supplementary Figure 4 Supplement 1 and 2 have been mistaken for each other – please readjust.

16. Supplementary Figure 4 (Figure supplement 1): In panel B apparently different protein concentrations have been used compared to panels A and C – why?

17. Supplementary Figure 4 (Figure supplement 2): Panels BC – Please indicate error bars and replicate numbers.

18. Figure 4A: Why don't the authors compare the reaction half-times of wild-type and the D51R mutant at similar concentrations to enable a more direct comparison between the two? Are the reaction half-times based on the kinetic traces seen in the respective supplementary Figure? If yes, why is there such a difference in the reaction half-time between the trace at 1 nM PIP5K in the supplement (roughly 160 seconds) and the 280 seconds stated in the main Figure? Why are the traces for higher PIP5K concentrations not included in the supplement?

19. Figure 2 and Figure 4: Why is the fluorescence of a membrane dye used to calibrate for PIP5K membrane density in Figure 2, whereas a more sensitive FCS-approach is used in Figure 4 to do the same? They should have likewise measured FCS for the Alexa-fusion construct in Figure 2.

20. Lines 294-301: in Line 296, the authors give a definition of the reaction coordinate x. However, they are not defining the omega factor that they are using, and it is not clear what they mean with PIP1 and PIP2 – should that be the educt and product? Please clarify and annotate in the text.

21. Lines 324-325: Please add the information that the DrrA-OCRL construct is the opposing 5`-phosphatase.

22. Figure 5B: What is meant with 0`-9`? Are these frame numbers (if yes what is the time difference between them? If the numbering should indicate time – what unit is it? Same question for Figure 6A).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Membrane-mediated dimerization potentiates PIP5K lipid kinase activity" for further consideration by eLife. Your revised article has been evaluated by Jonathan Cooper (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there is one remaining issue that needs to be addressed, as outlined below:

Regarding the text starting on line 375:

"Conversely, the phosphorylation rate constant for wild-type PIP5KB began with a slow rate and then transitioned to a rate of 2.7x10-3 lipids/μm2•sec per kinase as the reaction progressed."

However, when examining the plots in Figure 5G, it is not clear that there is a slow component to the rate. If the slower rate is in the region of the graph close to the origin, then this area should be highlighted and zoomed in with an inset plot showing an expanded scale. Otherwise, this statement is not corroborated with the data as shown.

https://doi.org/10.7554/eLife.73747.sa1

Author response

Essential revisions:

In general, the reviewers found this paper to be of high interest, presenting exciting results leading to a mechanistic understanding of regulation of pattern forming behavior by PIP5K kinase in membranes. While the data presented was mostly convincing, there are concerns about the interpretation of whether this is dimer specific or related to higher-order oligomerization. In addition, the study suffers from a lack of clarity in the writing, as well as inconsistencies and shortcomings in data presentation. Specifically, the following revisions are required:

1) While there is evidence of linkage to oligomerization, the overall interpretation that the mechanism is specifically linked dimerization is not strongly supported. To address this, the studies that are currently presented require additional clarification, and control experiments are necessary to map out the dimer specific signal on membranes. Alternatively, the mechanism may be modified to consider that this effect may come from an ensemble of higher-order oligomeric states. With this, the paper and the title require significant revisions to clarify this point.

Our revised manuscript contains a significant amount of new data demonstrating that fluorescently labeled PIP5K and PIP4K can bind to membranes as either monomers or dimers, respectively, at low surface densities. This is supported by control experiments analyzing the stepwise photobleaching of single membrane bound particles of mNG-PIP5K and mNG-PIP4K. Consistent with SEC data, our single molecule experiments do not show any evidence of high-order oligomers forming on supported membranes. In the Discussion section, we speculate that high-order oligomers can potentially form. However, there is currently no direct evidence to support the formation of high-ordered PIP5K oligomers. If such a mechanism exists, future structural biochemistry studies will be necessary to identify a new protein-protein binding interface that mediate this unknown interaction.

During our revision, we established new experimental conditions to directly visualize membrane mediated dimerization of mNG-PIP5KB monomers, while still being able to spatially resolve single molecules (see Figure 4). The small fraction of dimeric mNG-PIP5KB species observed on membranes is absent from the population when we characterize the dimerization deficient mutant, PIP5KB (D51R). The efforts to directly visualize mNG-PIP5KB were made possible by a graduate student, Benjamin Duewell. In our revised manuscript, Benjamin is now included as a contributing author.

The reviewers suggested that we modify the title of our manuscript to state that PIP5K activity is potentiated by higher order oligomerization of PIP5K, rather than dimerization. Based on our additional control data, we more convincingly demonstrate that membrane-mediated dimerization is the mechanism that enhances PIP5K lipid kinase activity. Hopefully our revisions clarify the issues raised in our original submission and that the reviewers agree with our overall conclusions.

2) Sample sizes, errors and statistical tests must be calculated and clearly presented in the paper and in the figure legends. In its current form, it is not possible to assess whether the results presented are reproducible.

Details about sample size and statistics were located in the Methods section of our original submission. In our revised manuscript, a more comprehensive description of sample size and statistics is included in the figure legends, Table 1, Table 2, and the main text.

3) The writing needs significant revisions to improve coherency and clarity. The manuscript should be revised to improve accessibility for a broad group of readers, with improved background and discussion of the physical concepts and equations that are presented. In addition, the discussion should be revised to better related the current findings to recently published studies.

We appreciate the reviewer comments that our writing needed improvement to maximize accessibility and readership. In our revision, we expanded descriptions of our methodology and rationale for different experiments. We describe the limitation of some approaches (e.g. FCS) and explain why an alternative approach was necessary. The addition of control data should also improve the clarity of our results and conclusions.

4) Address the specific comments and questions raised by the reviewers below.

We addressed all the review comments, point by point. In our rebuttal, we have also copied and pasted textual revisions below specific reviewer comments. For readability, minor typos and adjustments to figure numbering were corrected, but not highlighted in the main text.

Overall, the study would benefit from editing to improve coherency and clarity.

Reviewer #1 (Recommendations for the authors):

1. The results presented in the size exclusion chromatography analysis (Figure 1B) need some clarification. There is a hypothesized dimer peak for PIP4K, monomer peak for zPIP5KA/D84R and a sharp monodispersed peak for zPIP5KA, that is interpreted as a monomer/dimer mix. While rapid monomer-dimer exchange might account for this, it is surprising that such as mixture is not smeared over both monomer and dimer elution volumes. Changes in column behavior can also create the appearance of peak shifts and so additional calibration is necessary in order to be able to interpret this data. The protein samples should be run with a calibration sample that can control for changes in column behavior and also allow the identification of expected peak positions. In addition, the protein concentration prior to loading should be controlled, as this can also lead to observation of different oligomeric species. An alternate method that often presents clearer results is cross-linking the dilute protein sample by something like glutaraldehyde and the running on SDS-PAGE. This may be used to rule out higher-order oligomeric species.

The reviewer makes a good point concerning the limitations of our SEC analysis. We also agree that it’s surprising the zPIP5KA monomer/dimer mix is not distributed over a broad peak. The main purpose of the SEC data included in our manuscript was to confirm previously reported results from Hu et al. (2015), who reported a similar elution profile to ours. Since our SEC data is confirmation of published results and less conclusive compared to our single molecule imaging experiments, we moved the SEC data from Figure 1 to the Figure 1 —figure supplement 1 in our revised manuscript. To strengthen our SEC data, we ran several protein molecular weight standards over our Superdex 200 column, which we compare to the elution profiles of PIP4KB, zPIP5KA, and zPIP5KA(D84R). In addition, we purified more mNG-PIP5KB (wild type and D51R) and compared their elution profiles on a Superdex 200 column (Figure 4A). To rule out the presence of higher order oligomers we performed single molecule photobleaching experiments (Figure 1).

Overall, we conclude that wild type and dimerization deficient PIP5K mutants exhibit distinct SEC elution profiles. We and other groups have interpreted this result as representing either the monomeric or dimeric states of PIP5K. However, when mNG-PIP5K is diluted to a sub-nanomolar concentration and characterized by smTIRF microscopy, it appears to be exclusively monomeric based on our molecular brightness analysis. By contrast, when PIP4K is diluted to a sub-nanomolar concentration, it binds to supported membranes exclusively as a dimer. This supports a model in which PIP5K exists in a monomer-dimer equilibrium, while PIP4K is a constitutive dimer. Unfortunately, we and others have not been able to measure the K_D for PIP5K dimerization in solution. This challenge potentially reflects the weak affinity between PIP5K monomers.

2. The brightness analysis presented in Figure 1D,E indicates either that the mNG is folded and fluorescent with 100% yield, or that the PIP4KB being observed is a higher order oligomer than dimer. The PIP4KB intensity distribution in Figure 1E shows very little overlap with the monomeric distributions, and a broad intensity distribution that is 2-3 fold greater that the monomeric samples. Considering binomial labeling probabilities, with around 80% labeling efficiency, a dimer is expected to yield nearly equal probabilities of single and double labelled species, which does appear to be the case for the fluorophore labelled protein presented in Figure 1 —figure supplement 2. For the mNG samples, it is rare for the fluorophore to fold with 100% efficiency, and so it is more probable that the resultant intensity distribution corresponds to > 2 proteins. Photobleaching analysis of the spots would reveal clearer information and test whether higher-order oligomeric species are present.

This is a good point raised by the reviewer. We believe that part of the confusion concerning our interpretation of PIP4K/PIP5K oligomerization stemmed from the way in which data was originally plotted in Figure 1E (i.e. 3D plot with a tilted x-axis). We now provide more extensive experimentation showing that we only observe membrane bound monomers and dimers in our single molecule imaging experiments. Based on our additional characterization, there is no evidence supporting the formation of higher order PIP4K or PIP5K oligomers.

Based on our spectrophotometry measurements, mNG and mNG-PIP5KB purified from either bacteria or insect cells contain ~80% mature and fluorescent chromophores. This is calculated based on the fraction of purified mNG protein that absorbs light at 517 nm, compared to the total protein concentration calculated based on absorption at 280 nm. Knowing this, a protein that is constitutively dimeric (i.e. PIP4K) will exist as a population of molecules with either 1 or 2 mature mNG proteins when visualized using single molecule TIRF-M. Supporting this notion, we performed single molecule step-photobleaching experiments of mNG-PIP4KB (Figure 1D). By monitoring the maximum pixel intensity of single mNG-PIP4KB molecules, we observed that ~80% of the protein contain 2 mature mNG. We never observed mNG-PIP4KB particles photobleached in more than 2 steps (Figure 1D). By contrast, mNG-zPIP5KA, mNG-PIP5KA, and mNG-PIP5KB all photobleached in a single step (Figure 1E and Figure 1 —figure supplement 4). To quantify changes in molecular brightness during step-photobleaching, we continuously imaged single mNG-PIP4K and mNG-PIP5K particles. In Figure 1F-1G, we show how the distribution of mNG-PIP4K molecular brightness shifts from bimodal to unimodal over the course of the 10 second image acquisition. Conversely, the distribution of mNG-PIP5K molecular brightness remains unimodal because particles always photobleached in a single step. Plotted in Figure 1H, we show how the proportion of mNG-PIP4K and mNG-PIP5K with either 1 or 2 visible mNG molecules changes as a function of time from the beginning of image acquisition. Also see Video 1.

3. The statement of weak monomer-dimer equilibrium is not strongly supported by the presented data. To say this, one would need to show a reversible monomer to dimer conversion as a function of protein density, such as an association isotherm. While the data presented shows clear evidence of monomers, the change in behavior with protein concentration is in line with higher order oligomerization. This is even diagrammed in Figure 2A. To isolate the dimer-specific species signal, functional cross-linking of the protein might be useful to switch the protein from monomeric to dimeric states and characterize the dimer-specific signal at low protein densities.

Previously published research established that zPIP5KA is a dimer in solution based on size exclusion chromatography and multiangle light scattering (Hu et al. 2015). However, when we dilute zPIP5KA and other PIP5K family proteins to sub-nanomolar concentrations, mNG labeled kinases associate with PI(4,5)P₂ containing membranes primarily as monomers. This result implies that PIP5K must be in a weak monomer-dimer equilibrium. By contrast, dilution of mNG-PIP4KB to a sub-nanomolar concentration does not cause the kinase to dissociate into monomers. Based on our improved single molecule brightness analysis (see Figure 1), there no evidence of higher order oligomers according to the smTIRF measurements.

In our revised manuscript, we include a new Figure 4 showing the direct visualization of mNG-PIP5KB dimer formation on supported membranes.

In our revised manuscript, we added a comment in legend Figure 2A to emphasize that the cartoon is not drawn to imply a particular oligomerization state. We’re open to suggestions if the reviews have any ideas for illustrating an increase in protein surface density without implying a specific oligomerization state.

Chemical cross-linking experiments is a great idea. However, the rigorous biochemistry needed to validate the function of a crosslinked dimer and avoid artifacts associated with chemical crosslinking extend beyond the scope of this study.

4. The major argument for the effects being dimerization dependent are from the results of the D51R mutation, as it is interpreted that this mutation solely affects dimerization. However, it seems plausible that other things may change in the protein that impact catalytic activity that are outside of the dimerization. It is unclear as to whether this particular mutant is well folded on its own, thus further analysis of the protein would be useful here. Is the binding and catalytic activity preserved for D51R in solution, and how does this compare to wild-type under monomeric conditions? If dimerization was observable, for example via AUC, then this would also provide supportive evidence of the fold, as well as report the change in stability.

Previous biochemical analysis of zPIP5KA using SEC/MALS showed that this lipid kinase can form a dimer in solution, while zPIP5KA (D84R) was an apparent monomer by SEC/MALS (Hu et al. 2015, Nature Comm., doi.org/10.1038/ncomms9205). In our hands, we observe the same SEC elution profile for zPIP5KA (wild type and D84R) that was reported by Hu et al. (2015) (see Figure 1 —figure supplement 1). Hu et al. (2015) also previously showed by circular dichroism that mutations that disrupt the zPIP5KA dimer interface do not change the α helical character of the kinase compared to the wild type enzyme. This suggests that the kinase is folded properly.

Using smTIRF, we show that Ax647-PIP5KB(D51R) at a low membrane surface density still binds cooperatively to PIP₂ lipids (Figure 3D). Dwell time analysis of both PIP5KB (WT and D51R) yielded indistinguishable results (Figure 3C-3D and Table 2). This indicates that the D51R mutant is folded properly, because misfolding would disrupt membrane docking and association with PIP₂ lipids.

Another strong indication that the D51R mutation only disrupts dimerization is our ability to introduce a structural compensatory mutation (D51R/R254D), which fully restores kinase activity (Figure 5A-5D). If the D51R mutation caused misfolding, the lipid kinase activity would still be reduced in the D51R/R254D mutant.

Throughout the course of this study, we empirically determined that mutations that disrupt protein folding cause PIP5K to be proteolyzed during expression and purification from insect cells. Prior to the Hu et al. (2015) zPIP5KA crystal structure, Lacalle et al. (2015, FASEB Journal, doi: 10.1096/fj.14-264606) reported that they determined the dimer interface of PIP5K based on a structural homology model built from PIP4K. However, in light of the Hu et al. (2015) structure paper, it’s clear that the Lacalle et al. model for PIP5K dimerization is incorrect. Prior to this knowledge, however, we purified the following mutants from insect cells based on Lacalle et al. (2015) PIP5K dimerization model: F190A/L191A/P192A and L35A/Y39A/N43A. Both of these reported “dimer interface” mutants were unstable and proteolyzed during the purification, presumably due to protein misfolding (data not shown). We were never able to biochemically characterize any of “dimer interface” mutants reported by Lacalle et al. (2015). However, the Hu et al. (2015) crystal structure of zPIP5KA, allowed us to successfully purify the following dimer mutants from insect cells without any proteolysis issues: zPIP5KA (D84R), hPIP5KB (D51R), and mPIP5KA (D92R).

5. The binding curves for PIP5KA to the membrane in Figure 2F are very clear and present interesting results on the different binding capacities as a function of PIP2. Since the lower leg of the monomer-oligomer equilibrium reaction on the membrane (Figure 4D) will be dependent on the membrane associated protein density, it is important to have this full binding relationship for all constructs in order to interpret the results from the different experiments. As such, this information seems essential for the interpretation of the D51R data. While the dwell time and diffusion distributions show similar behavior to WT, this only reports on the behavior of the molecules that are bound and are all likely to be monomers under the examined conditions (Figure 3C,D). It does not directly report on whether there is the same amount of D51R vs. WT on the membrane. In this case, the data presented in Figure 5, is not expected to have the same amount of membrane bound PIP5K, which could account for the differences in behavior rather than dimerization. Indeed, the addition of 1 μm of D51R increases the observation of the compositional patterns.

We thank for the reviewer for this excellent observation. In our revision, we include a plot that shows the relationship between mNG-PIP5KB solution concentration and membrane binding intensity for both wild type and dimer interface mutant (D51R) (Figure 5F). In addition, we reconstitute the bistable spatial patterning reaction in the presence of mNG-labeled PIP5K (wild type and D51R). We find that PIP lipid compositional pattern created by PIP5KB (D51R) contain 2x more kinase bound to the membrane compared to the wild type kinase (Figure 6B-6D). However, the reduced activity of the dimer mutant prevents mutant kinase from robustly competing against the opposing lipid phosphatase to form a stable domain of PI(4,5)P₂ lipids.

Reviewer #2 (Recommendations for the authors):

1. It is not a weakness but rather a limitation that the study is not extended experimentally into real cell situations and only speculates about the potential impact of its finding to the control of the enzymes in real cells.

We agree and look forward to performing complementary experiments in vivo to better understand the mechanisms controlling plasma membrane recruitment of PIP5K in the future.

2. Some of the methods and terms should be defined and explained for the general reader: e.g. It would be useful to define "dwell time" or explain what the Y axis is in Figure 2B. (for example what is CDF?).

In our revision, we defined terminology related to our single molecule experiments. For example, CDF is an abbreviation for cumulative distribution frequency. As suggested by the reviewer, we have defined abbreviations in the main text and figure legends. Additional discussion of related terms is also included in the methods section.

3. Similarly, the detailed description of how the catalytic activity of the enzymes were calculated (lines 270-310) is important for the experts. However, it would be desirable to "translate" the following sentences to the general reader.

"When the derivative of the PIP5K kinase reaction traces were plotted against the reaction coordinate, x, the curve displayed a high degree of asymmetry that required a second-order term to fit (kWT(x) = k0 + k1x + k2x2) (Figure 4E). In contrast, the dx⁄dt curve for the dimer mutant was parabolic and could be fit using an equation that describes an enzyme with first order positive feedback (kD51R(x) = k0 + k1x) based on product binding (Figure 4E)." This may not be necessary if the paper is published in a biophysical journal, but for most of the readers of eLife, this description is not very accessible.

We thank the reviewer for point out how our writing is less accessible to most eLife readers. We have replaced some of the jargon with terms that communicate a more simple and clear explanation.

4. The legend to Figure 4 Supplement 1. does not really describe what is measured here. First of all, what is measured as "auto-correlation" should be explained. It is not clear what the different colors mean (the concentrations refer to what component?). Panel A-C appear to show graphs on the various PIP5KB forms (wt or mutants), but the Figure legend for panel A does not refer to PIP5KB.

5. The same is true for the legend to Figure 4 Supplement 2. From the description, one would expect to see three similar measurements for the three forms of the enzyme. Clearly, panel A shows a curve that is not explained. Panels B and C show FCS density as a function of TIRF intensity. What is measured here (what is FCS density?)? How do these curves inform about lipid kinase activity? Clearly, the Authors should do better to explain what is shown here and how does that translate to kinase activity.

We apologize for the error, which caused confusion. In our original submission, figure legends for Figure 4 —figure supplement 1 and 2 were swapped. We have corrected this error. Additional text has been added to define and explain the meaning of auto-correlation.

6. I think the DrrA designation of what is better known as SidM should at least be clearly stated somewhere (preferably also in the Figure legends).

In the methods sections, we note that this PI(4)P lipid binding domain, DrrA/SidM (544-647aa), is derived from Legionella pneomophila. When this PI(4)P sensor is first introduced in legend Figure 6, we now mention that DrrA is also known as SidM in L. pneomophila. To be consistent with our previous publication (Hansen et al. 2019 PNAS), we refer this PI(4)P lipid binding domain as DrrA throughout the manuscript. We have also included an expended description in the methods section titled, “Molecular Biology.” The following statement has been added to the main text of the manuscript when DrrA is first mentioned:

“To test this hypothesis, we compared the ability of PIP5KB and the PIP5KB (D51R) dimer interface mutant to form bistable PIP compositional patterns in the presence of an opposing 5-phosphatase with engineering positive feedback based. This was achieved by fusing a minimal PI(4)P binding motif, referred to as DrrA (or SidM/DrrA), to OCRL (Hammond, Machner, and Balla 2014; Zhu et al. 2010). The resulting chimeric 5-phosphatase, DrrA-OCRL, was previously shown to exhibit positive feedback based on PI(4)P recognition (S. D. Hansen et al. 2019).”

7. Remove the prime from all "5'-phosphatase". There are no prime numbers on the inositol ring just simple numbers designating the positions on the single carbon ring.

Thank you for the feedback. We removed all the prime numbers when mentioning 5-phosphatase in the manuscript.

Reviewer #3 (Recommendations for the authors):

1. Line 150 and Figure 1B: The authors are referring to a dimer mutant of PIP5K. They need to clarify if that means that the protein is locked in its dimerized state or if the dimerization interface is disrupted and thus the protein resides in its monomeric state. If, as indicated by the 'dimer mutant' reference the protein should be locked in a dimer state, why does the SEC elution profile and their annotation rather indicate that the protein elutes as a monomer? Also, it would be beneficial for the reader to indicate the name of the dimer mutant in the text.

All dimer mutants presented in our study disrupt the dimer interface resulting in a monomeric form of PIP5K. The dimer interface mutations are based on the published crystal structure and biochemical characterization of zebrafish PIP5KA (Hu et al. 2015, Figure 3A). In Figure 3B we show a sequence alignment and highlight the specific aspartic acid and arginine residues that form a salt bridge that stabilizes the dimer interface. Building off the work by Hu et al. (2015, Nature Communications) we characterized the functional significance of PIP5K dimerization on membranes and role it serves in regulating catalysis.

2. Figure 1 Supplement 2: Monomer/dimer distribution of mNG-PIP4K in Figure 1E are considerably different to the ones shown for the Alexa488-PIP4K version in supplement 2. Is there a reason for this comparison, especially as the authors are later introducing another chemical label with the Alexa647-conjugation construct? They should rather repeat the experiment with the Alexa647-conjugation construct that they are using later on.

The difference in molecular brightness distributions between Figure 1F-G and Figure 1—figure supplement 3 (note updated figure numbers) reflects the difference in labeling strategies and their respective efficiencies. The main point of this supplemental figure was to emphasize that PIP4KB and PIP5KB can be labeled with different fluorescent probes and we still observe a difference in their molecular brightness distribution. We include this control to emphasize that the difference in distributions shown in Figure 1F-1G is not solely dependent on using mNeonGreen.

In this study, we did not quantify the molecular brightness distribution of Ax647-PIP4KB or Ax647-PIP5KB. Although labeling with Alexa647 results in a very photostable fluorescent protein (see Figure 2—figure supplement 1 for photobleaching kinetics), the labeling efficiency achieved using Sortase mediated peptide ligation is 25-45% in the presence of Alexa647. We consistently observe this low labeling efficiency independent of the specific protein being labeled. We believe that Sortase generally has a difficult time using the Alexa647-LPETGG peptide as a substrate. Interestingly, Alexa488-LPETGG results in a higher labeled efficiency (50-90%) for all proteins we’ve labeled to date. However, the brightness and photostability of Alexa488 is comparable to mNeonGreen. In this study, we found that mNG kinase fusions were the more reliable option for measuring molecular brightness, because we consistently obtain ~80% mature and fluorescently labeled enzyme. Our revised manuscript notes the measured labeling efficiency in the figure legend. We also include the following discussion the main text of the manuscript concerning the important point raised by the reviewer:

“We also observed similar differences in molecular brightness comparing recombinantly expressed and purified PIP4KB and PIP5KB that were chemically labeled with Alexa488 in vitro using Sortase mediated peptide ligation (Figure 1—figure supplement 3). However, the lower labeling efficiency achieved using Sortase mediated peptide ligation, led us to measure the molecular brightness distribution of PIP5K homologs and paralogs using exclusively mNG fusion proteins in this study.”

3. Figure 1B: In Figure 1B, the 'dimer' mutant of PIP5K is annotated as zPIP5KA(D84R), however in the respective figure caption, it is referred to as zPIP5KA(D51R) mutant – which one now is the correct mutant? The authors should use a size standard to calibrate the SEC elution profiles to ensure the correct detection of monomers and dimers (SEC-MALS would be even better suited). The critical reader would furthermore like to see an SDS-PAGE gel of the purified proteins, as especially the elution profile of PIP5K indicates impurities.

We corrected this typo to indicate the correct dimer interface mutation of D84R for zPIP5KA. For reference, Figure 3B shows a sequence alignment of PIP5K protein family members with the amino acid side chains that stabilize the dimer interface.

In our revised manuscript, we include data showing the calibration of our Superdex 200 column using a variety of protein molecular weight standards (Figure 1—figure supplement 1). In legend Figure 1—figure supplement 1, we note that the elution profile represents the final step in the zPIP5KA protein purification. We also indicate the presence of small molecular weight impurities in the SEC elution profiles using asterisks (Figure 1—figure supplement 1C).

In our revised manuscript, we also include new a SEC elution profile for purified mNG-PIP5KB and mNG-PIP5KB (D51R) in Figure 4A. The elution volume for protein molecular weight standards are indicated in the graph.

Our typically yield of 0.2 mg total protein from 4 liters of insect cells prohibiting extensive characterization of oligomerization using SEC. Given that SEC alone does not report absolute molecular weight of protein complexes, we prioritized the more definitive single molecule TIRF experiments to characterize the oligomerization state and dynamics of membrane bound mNG-PIP4K and mNG-PIP5K.

4. Figure 1C/D: The authors should indicate the abbreviations of both proteins in the respective figure captions.

In the main text, we include the following statement:

“Comparing the molecular brightness distributions of mNG-PIP5KA (mouse), mNG-PIP5KB (human), mNG-PIP5KC (human), mNG-zPIP5KA (zebrafish), and mNG-yMss4 (yeast multicopy suppressor of Stt4 or yeast phosphatidylinositol 4-phosphate 5-kinase), we observed only single peaks corresponding to monomeric kinases labeled with a single fluorescent mNG (Figure 1I).”

In Figure legend 1, we define abbreviation shown in Figure 1 as follows:

“Molecular brightness frequency distribution plots measured in the presence of mNG tagged mouse PIP5KA (5KA), human PIP5KB (5KB), human PIP5KC (5KC), zebrafish PIP5KA (z5KA), yeast Mss4 (yMss4), and human PIP4KB (4KB).”

5. How do the authors account for bleaching in their TIRF single particle tracking assay?

In our revised manuscript, we report the bleaching kinetics of surface immobilized Alexa647 labeled PIP5K (t _{bleaching, Ax647-5K} = 26.7 seconds). This data is reported in the main text, Figure 2 legend, and plotted in Figure 2 —figure supplement 1. Rather than correcting all our dwell time measurement, we note that the bleaching kinetics are significantly long compared to the single molecule membrane interactions.

6. Supplementary Figure 1A: What is the reproducibility of the mNG-calibration curve for the quantitative analysis of kinase presence in the cell lysate? As indicated in the figure caption, only a representative curve is shown, however adding standard errors for each concentration and the indication of the number of replicates would need to be included.

In our revised manuscript, three mNG-calibration curves are now shown in Figure 1 —figure supplement 2. Although our fluorescent protein plate reader measurements are pretty consistent day-to-day, for these measurements we generate a new mNG-calibration curve every time we measure the concentration of mNG-PIP5K in cell lysate. This is similar to creating a BSA standard curve every time a Bradford assay is performed. As a result, adding standard error bars for each concentration was unnecessary.

7. Lines 180-181: Several mNG-fusion constructs are named, but it is completely unclear for the reader what they are, as they have not been introduced (e.g. mNG-PIP5KA/B/C or mNG-yMss4?). Even more importantly, in Figure 1E, which is the figure that has been referenced in the text, there are again other abbreviations for the used constructs that do not fit the ones named in the text or have not been referenced in the text. Likewise, also no consistency between the abbreviations between Figures 1E and F. the authors need to clarify this and keep the names of constructs consistent throughout the whole text, figures, and supplementary information.

We thank the reviewer for recognizing the inconsistencies in our naming of the PIP5K paralogs and homologs. We have corrected the inconsistencies found in Figure 1 and throughout the main text, figures, and supplementary information.

In the main text, we include the following statement:

In Figure legend 1, we define abbreviation shown in Figure 1 as follows:

8. Figure 1F: The authors are referring to a step size distribution in the figure caption, but at the same time state diffusion coefficients for the respective constructs – they need to elaborate on how exactly they got the diffusion coefficients from the step size distribution, more specifically: what kind of fitting they used. As in Figure 2C, the fit should also be included in the plot. Furthermore, what is the error and standard deviation for the diffusion coefficients? It would be beneficial for the reader to also state the deviations in lines 190/191.

In our original submission, the method used to fit the step size distribution was described in the methods section titled, “Single particle tracking.” Below are the equations used to fit the distributions throughout the manuscript:

Single species model:

$f (r) = \frac{r}{2 D τ} e^{- (\frac{r^{2}}{4 D τ})}$ Two species model: $f (r) = α \frac{r}{2 D_{1} τ} e^{- (\frac{r^{2}}{4 D_{1} τ})} + (1 - α) \frac{r}{2 D_{2} τ} e^{- (\frac{r^{2}}{4 D_{2} τ})}$ In the case of Figure 2C, the dashed black line represents the curve fit of the step size distribution. In our revised manuscript, we include the appropriate statistics and errors associated with our diffusion coefficient measurements. In Figure legend 2C, we state that the dashed black line represents the curve fit used to calculate the diffusion coefficient.

9. As stated in line 192, the authors are comparing the diffusion coefficients of the cell lysate kinases with purified kinases in Figure 2C. However, in the two cases different membrane compositions have been used – in Figure 1F they state that 2 % PIP2 have been used, while in Figure 2C 4 % have been incorporated. Why do they change the proportion of PIP2 in the membrane between the two cases? This inconsistency especially impedes their assumption in line 193, that the membrane binding of kinases in the cell lysate is indistinguishable from the purified case.

Thank you for pointing out that we changed two variables when making the comparison between the mNG-PIP5K in lysate and purified proteins. To clarify, the data in Figure 1 was collected using SLBs with 4% PI(4,5)P₂, while the membranes in Figure 2 contain 2% PI(4,5)P₂. The rationale for lowering the 2% PI(4,5)P₂ was mainly to observe the protein density dependent change in Ax647-PIP5KB membrane dwell time (Figure 2B) without encountering problems associated with photobleaching and underestimating the change in the dwell times.

In our revised manuscript, we provide broader characterization of PIP5K membrane diffusivity. Our conclusion, is that diffusivity of PIP5K is not significantly different in the presence of 2% versus 4% PI(4,5)P₂. In addition, mNG-PIP5K visualized dilute cell lysate also behave similar to purified mNG-PIP5K. Please refer to Table 1, Table 2, and Figure 3 —figure supplement 1 for additional quantification and statistics from single particle tracking data.

10. Figure 2B: For readers that are not familiar with single particle tracking, the cumulative density function should be introduced in the caption of the figure or even in the axis label. Same comment for Figure 3CDE.

We have included a more accessible description of cumulative distribution frequency (CDF).

The methods section includes the following description:

“To calculate the single molecule dwell times for Ax647-PIP4K and Ax647-PIP5K we generated a cumulative distribution frequency (CDF) plot using the 50 ms frame interval as the bin size. The log₁₀(1-CDF) was then plotted against the dwell time and fit to either a single or double exponential decay curve.”

We included the following statement in Figure Legend 2B:

“Ax647-PIP5K dwell times were calculated by fitting log₁₀(1-cumulative distribution frequency (CDF)) to either a single or double exponential decay curve (dashed black lines). Bin size equals 50 ms. See Table 1 for values and statistics.”

11. Figure 2C: Again, a new abbreviation is being introduced for PIP5KB.

We have corrected the abbreviation in Figure 2C to be more consistent throughout the manuscript.

12. Figure 2F: Error bars are missing and SEM of Kd values. In the respective caption, they should state the number of independent replicates. They also need to indicate in the caption that the nh – values they are referring to in the figure are the Hill coefficients. The manuscript would benefit considerably if they would repeat the experiments shown in this Figure also with the D51R mutant – especially as they are comparing the mutant with the wild type throughout the manuscript, and only for Kd values the respective differences cannot be judged. Similar comment for Figure 4AB regarding error bars and replicate numbers.

We have included the statistics and additional information of the curve fitting in Figure 2F. In the main text, we refer to the curving model as the “concerted model for cooperativity,” which is the terminology used to describe the Hill equation. We added the following text to Figure legend 2:

“Lines represent curve fit using concerted model for cooperativity (i.e. Hill equation). n_H is the Hill coefficient. Points are mean values [N = 15-20 fluorescent intensity measurements per sample from 1 technical replicate].”

As suggested by the reviewer, we provide new experimental data comparing the membrane binding behavior of mNG-PIP5KB and mNG-PIP5KB (D51R). At saturating concentrations, we observed a 2-fold difference in protein surface density comparing mNG-PIP5KB and mNG-PIP5KB (D51R). This is consistent with a model proposed by Amos et al. (2019, Structure, doi.org/10.1016/j.str.2019.05.004) that is based on molecular dynamic simulations that showed only a single subunit of a PIP5KA dimer can bind to the membrane at a time.

The kinetic data in Figure 4 (know called Figure 5) was significantly updated to include the proper statistics. The following text can be found in Figure legend 5:

“(A-C) Representative kinetic traces monitoring the production of PI(4,5)P₂ in the presence of 1-5 nM PIP5KB, PIP5KB (D51R), or PIP5KB (D51R/R254D). The PI(4,5)P₂ membrane surface density was estimated based on the membrane localization of 20 nM Ax488-PLCd. Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Quantification of reaction half-time from trajectories shown in (A-C). Bars equal mean values [N = 2-4 reactions per concentration, error = SD].”

13. Figure 3CD: The authors need to indicate in the caption or the figure that this is a low protein density condition.

Figure legend 3 has been revised to state:

“(C-D) Dimerization is not required for cooperative PI(4,5)P₂ binding. Representative single molecule dwell time distributions measured at low protein density in the presence of either (C) 1-5 pM Ax647-PIP5KB and (D) 1-5 pM Ax647-PIP5KB (D51R).”

14. Figure 3F: Please state the respective diffusion coefficients you have obtained from the used fit. Same comment for Supplementary Figure 3 (and adjust x-axis label to be consistent with similar graphs).

The diffusion coefficients, along with statistics (i.e. number of trajectories, number of steps, and error) are included in both the figure legends and newly added Table 1 and Table 2.

15. Supplementary Figure 4 (Figure supplement 1 and 2): It seems like the Figure captions of Supplementary Figure 4 Supplement 1 and 2 have been mistaken for each other – please readjust.

We apologize for the error. We accidently swapped figure legends for Figure 4—figure supplement 1 and 2 in our original manuscript.

16. Supplementary Figure 4 (Figure supplement 1): In panel B apparently different protein concentrations have been used compared to panels A and C – why?

We originally performed lipid phosphorylation reactions at different concentrations of PIP5K (WT and D51R) because the proteins have different levels of activity. In our revised manuscript, we include kinetic traces for the lipid phosphorylation reactions, as well as the reaction half-times (Figure 5A-5D). All experiments have been performed at similar concentrations for wild type and mutant PIP5KB to more easily compare the reaction half-times.

17. Supplementary Figure 4 (Figure supplement 2): Panels BC – Please indicate error bars and replicate numbers.

We repeated these experiments comparing similar protein concentrations. The standard deviation for our reaction half-time measurements are now plotted in Figure 5D.

18. Figure 4A: Why don't the authors compare the reaction half-times of wild-type and the D51R mutant at similar concentrations to enable a more direct comparison between the two? Are the reaction half-times based on the kinetic traces seen in the respective supplementary Figure? If yes, why is there such a difference in the reaction half-time between the trace at 1 nM PIP5K in the supplement (roughly 160 seconds) and the 280 seconds stated in the main Figure? Why are the traces for higher PIP5K concentrations not included in the supplement?

Thank you for identifying this poorly presented data. In our revision, we include data that directly compares reaction kinetics of lipid phosphorylation in presence of PIP5K, wild type and mutants, measured at the same protein concentrations (Figure 5A-5C). Quantification of the reaction half-times for multiple kinetic traces is now included in Figure 5D.

19. Figure 2 and Figure 4: Why is the fluorescence of a membrane dye used to calibrate for PIP5K membrane density in Figure 2, whereas a more sensitive FCS-approach is used in Figure 4 to do the same? They should have likewise measured FCS for the Alexa-fusion construct in Figure 2.

In our manuscript, we present two complementary approaches to quantify the surface density of PIP5K. The use of a fluorescently membrane dye is an established method for quantifying membrane surface densities (see Galush, Nye, and Groves 2008). It has similar sensitivity to FCS measurements.

The slow diffusivity of Ax647-PIP5KB and mNG-PIP5KB prevented us from directly performing FCS measurements on membrane bound fluorescently labeled PIP5KB. Instead, the FCS data included in our manuscript is a measure of mNG attached to a supported lipid bilayer. The fluorescence intensity and membrane density of mNG was then used to calibrate the fluorescence intensity of other mNG fusion proteins (e.g. mNG-PIP5KB).

Due to limitation in instrumentation, we were unable to perform additional FSC measurements during the period of the manuscript revision. However, we don’t believe this is critical for interpreting our data and it does not change the conclusions of our manuscript.

20. Lines 294-301: in Line 296, the authors give a definition of the reaction coordinate x. However, they are not defining the omega factor that they are using, and it is not clear what they mean with PIP1 and PIP2 – should that be the educt and product? Please clarify and annotate in the text.

The omega factor refers to the fraction of either PIP1 or PIP2. For clarity, PIP1 and PIP2 have been redefined in the text to be PI(4)P and PI(4,5)P2, respectively. The following text was added to the main text:

“To analyze the feedback profiles, we plotted the rate of PI(4,5)P₂ production, $\frac{dx}{dt}$ , as a function of the reaction coordinate, x, as $x \equiv σ P I (4, 5) P 2 / (σ P I (4, 5) P 2 + σ P I (4) P)$ . $σ$ denotes the membrane density of each PIP lipid species throughout the entire reaction trajectory.”

21. Lines 324-325: Please add the information that the DrrA-OCRL construct is the opposing 5`-phosphatase.

“To test this hypothesis, we compared the ability of PIP5KB and the PIP5KB (D51R) dimer interface mutant to form bistable PIP compositional patterns in the presence of an opposing 5-phosphatase with engineering positive feedback based. This was achieved by fusing a minimal PI(4)P binding motif, referred to as DrrA (or SidM/DrrA), to OCRL (Hammond, Machner, and Balla 2014; Zhu et al. 2010). The resulting chimeric 5-phosphatase, DrrA-OCRL, was previously shown to exhibit positive feedback based on PI(4)P recognition (S. D. Hansen et al. 2019).”

22. Figure 5B: What is meant with 0`-9`? Are these frame numbers (if yes what is the time difference between them? If the numbering should indicate time – what unit is it? Same question for Figure 6A).

We apologize for the confusing units. In our revised manuscript, we clarified the units to indicate time in minutes.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there is one remaining issue that needs to be addressed, as outlined below:

Regarding the text starting on line 375:

"Conversely, the phosphorylation rate constant for wild-type PIP5KB began with a slow rate and then transitioned to a rate of 2.7x10-3 lipids/μm2•sec per kinase as the reaction progressed."

However, when examining the plots in Figure 5G, it is not clear that there is a slow component to the rate. If the slower rate is in the region of the graph close to the origin, then this area should be highlighted and zoomed in with an inset plot showing an expanded scale. Otherwise, this statement is not corroborated with the data as shown.

Thank you for the feedback concerning Figure 5G. This is a challenging piece of data to present. Based on your comment, we have modified Figure 5G to more clearly explain the transition in reaction kinetics shown in this plot. In addition, we have modified the main text and Figure Legend 5 to more clearly explain these results. An updated version of the manuscript and Figure 5 has been uploaded to reflect the new changes.

Main text edits (lines 375-381):

“Conversely, the phosphorylation rate constant for wild-type PIP5KB began with a slow rate and then transitioned to a rate of 2.7x10-3 lipids/μm2•sec per kinase as the reaction progressed (Figure 5G, see arrows indicating the transition from slow to fast kinetics for wild type PIP5KB). After the reaction reached the maximum velocity, the kinetics gradually slowed down due to substrate depletion. Overall, the difference in per-molecule reaction kinetics comparing PIP5KB and PIP5KB (D51R) was consistent with dimerization enhancing lipid kinase activity (Figure 5E), and establishes a positive feedback mechanism.”

Figure legend 5G edits (lines 755-762):

“…(G) Membrane-mediated dimerization enhances PIP5KB catalytic efficiency independent of membrane localization. Membrane localization of mNG-PIP5KB (WT and D51R) and production of PI(4,5)P2 were simultaneously monitored on supported membranes. Each data point in the plot represents the instantaneous velocity expressed as the number of PI(4,5)P2 lipids generated per μm2 per second per kinase as a function of the substrate density. The reaction catalyzed by PIP5KB displays the following three phases: (1) slow kinetics at reaction start, (2) rise to maximum reaction velocity, and (3) gradual decline in reaction velocity due to substrate depletion. (H)…”

https://doi.org/10.7554/eLife.73747.sa2

Article and author information

Author details

Scott D Hansen
1. Department of Chemistry and Biochemistry, University of Oregon, Eugene, United States
2. Institute of Molecular Biology, University of Oregon, Eugene, United States
Contribution
Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

For correspondence
shansen5@uoregon.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0001-7005-6200
Albert A Lee
1. Department of Chemistry, University of California, Berkeley, Berkeley, United States
2. California Institute for Quantitative Biosciences, Berkeley, United States
3. Department of Molecular and Cell Biology, Berkeley, United States
Contribution
Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing

Competing interests
No competing interests declared
Benjamin R Duewell
1. Department of Chemistry and Biochemistry, University of Oregon, Eugene, United States
2. Institute of Molecular Biology, University of Oregon, Eugene, United States
Contribution
Resources, Investigation, Methodology, Writing – review and editing

Competing interests
No competing interests declared
Jay T Groves
1. Department of Chemistry, University of California, Berkeley, Berkeley, United States
2. California Institute for Quantitative Biosciences, Berkeley, United States
Contribution
Conceptualization, Supervision, Funding acquisition, Writing – review and editing

For correspondence
JTGroves@lbl.gov

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-3037-5220

Funding

National Science Foundation (CAREER MCB-2048060)

Scott D Hansen

University of Oregon, Department of Chemistry and Biochemistry (lab startup funds)

Scott D Hansen

Novo Nordisk Foundation Challenge Programme (Center for Geometrically Engineered Cellular Systems)

Jay T Groves

National Institute of Health, NIGMS (National Research Service Award (NRSA))

Scott D Hansen

National Institute of Health, NIGMS (T32 GM007759)

Benjamin R Duewell

National Institute of Health, NIGMS (F32 GM111010-02)

Scott D Hansen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank members of the Hansen lab for critical reading of our manuscript. Funding was provided by NIH grant P01 AI091580 (JTG), by the Novo Nordisk Foundation Challenge Programme as part of the Center for Geometrically Engineered Cellular Systems (JTG), National Research Service Award Postdoctoral Fellowship (SDH, F32 GM111010-02), University of Oregon Start-up funds (SDH), NSF CAREER Award (SDH, MCB-2048060), and Molecular Biology and Biophysics Training Program (BRD, NIH T32 GM007759).

Senior Editor

Jonathan A Cooper, Fred Hutchinson Cancer Research Center, United States

Reviewing Editor

Janice L Robertson, Washington University in St Louis, United States

Reviewer

Janice L Robertson, Washington University in St Louis, United States

Publication history

Received: September 9, 2021
Preprint posted: September 22, 2021 (view preprint)
Accepted: August 16, 2022
Accepted Manuscript published: August 17, 2022 (version 1)
Version of Record published: September 13, 2022 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.