Condensation of Ede1 promotes the initiation of endocytosis

  1. Mateusz Kozak
  2. Marko Kaksonen  Is a corresponding author
  1. Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Switzerland

Abstract

Clathrin-mediated endocytosis is initiated by a network of weakly interacting proteins through a poorly understood mechanism. Ede1, the yeast homolog of mammalian Eps15, is an early-arriving endocytic protein and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and endocytic uptake is decreased. We show that in yeast cells, cytosolic concentration of Ede1 is buffered at a critical level. Excess amounts of Ede1 form large condensates which recruit other endocytic proteins and exhibit properties of phase-separated liquid droplets. We demonstrate that the central region of Ede1, containing a coiled-coil and a prion-like region, is essential for both the condensate formation and the function of Ede1 in endocytosis. The functionality of Ede1 mutants lacking the central region can be partially rescued by an insertion of heterologous prion-like domains. Conversely, fusion of a heterologous lipid-binding domain with the central region of Ede1 can promote clustering into stable plasma membrane domains. We propose that the ability of Ede1 to form condensed networks supports the clustering of early endocytic proteins and promotes the initiation of endocytosis.

Editor's evaluation

This article demonstrates that the early arriving endocytic protein Ede1, the yeast Eps15 homolog, can generate a separate liquid-phase in vivo when overexpressed and that the domains conferring this property are required and sufficient to nucleate endocytic patches. The genetic and microscopy results are compelling and they support the model of liquid-phase separation in endocytosis, even though alternatives are discussed.

https://doi.org/10.7554/eLife.72865.sa0

Introduction

Clathrin-mediated endocytosis is a process which eukaryotic cells use to produce small transport vesicles from their plasma membrane. These vesicles deliver cargo molecules to the endolysosomal trafficking network where they are sorted for recycling or degradation (Grant and Donaldson, 2009). This process is the primary route of internalization of extracellular and surface molecules in eukaryotic cells (Kirchhausen et al., 2014).

Clathrin-mediated endocytosis requires a complex protein machinery to assemble on the plasma membrane in a specific sequence (Kaksonen et al., 2005; Sirotkin et al., 2010; Taylor et al., 2011). Endocytosis starts with the arrival of pioneer proteins which select the site and initiate the assembly of the endocytic coat (Kaksonen and Roux, 2018). Different pioneer proteins (Henne et al., 2010; Cocucci et al., 2012; Ma et al., 2016), lipids (Antonescu et al., 2011) and cargo molecules (Liu et al., 2010; Layton et al., 2011) have been shown to promote the initiation step (reviewed by Godlee and Kaksonen, 2013). However, the exact mechanism by which the endocytic sites are initiated remains poorly understood.

The molecular mechanisms of clathrin-mediated endocytosis have been studied extensively in yeasts, such as the budding yeast Saccharomyces cerevisiae. The pioneer module in yeast includes highly conserved adaptor proteins which bind membrane and cargo, such as the adaptor protein complex 2 (AP-2 complex), Syp1 (FCHo1/2 in mammals) and Yap1801/2 (AP180). Two conserved scaffold proteins, clathrin and Ede1 (Eps15), also arrive during the early phase. In contrast to the remarkably well-ordered assembly of the membrane-bending phase (Picco et al., 2015), the early proteins lack a specific sequence of recruitment (Carroll et al., 2012; Pedersen et al., 2020). Curiously, all the genes coding for the earliest-arriving proteins can be deleted without completely blocking endocytosis (Brach et al., 2014). However, the frequency of endocytic events is decreased in such cells, and the ability to regulate cargo recruitment is drastically compromised.

Ede1, a homologue of mammalian Eps15, is one of the key early proteins. It is among the earliest to appear at the nascent endocytic site (Carroll et al., 2012). The deletion of the EDE1 gene reduces the overall membrane uptake by 35 % (Gagny et al., 2000) and decreases the number of productive ndocytic events by 50 % (Carroll et al., 2012; Kaksonen et al., 2005). Ede1 interacts via its three Eps15 homology (EH) domains and other interaction motifs with proteins such as the AP-2 complex, Hrr25, epsins, Sla2, Syp1, and Yap1801/2 (Maldonado-Báez et al., 2008; Reider et al., 2009; Peng et al., 2015). Several of these proteins depend on interaction with Ede1 to become enriched at the endocytic sites (Carroll et al., 2012). Ede1 also oligomerizes via its coiled-coil domain, which is required for it to properly localise and function (Boeke et al., 2014; Lu and Drubin, 2017).

In recent years, liquid-liquid phase separation of biomolecules has garnered much attention as a mechanism for assembly of cellular organelles which lack surrounding membranes, such as P granules, nucleoli and stress granules (Banani et al., 2017). Such membrane-less compartments can accelerate reactions, sequester molecules from the cytoplasm or establish spatial organisation (Lyon et al., 2021). The phase separation framework can also apply to sub-micrometre compartments that balance the needs of concentrating select components with allowing a dynamic exchange and rearrangement of molecules. Examples of such compartments include transcriptional superenhancers (Sabari et al., 2018) and a wide array of membrane receptor clusters (Case et al., 2019).

In this work, we show that Ede1 has a propensity to form cellular condensates. We demonstrate that the cytosolic concentration of Ede1 is buffered at a critical concentration. We identify the molecular features driving the condensation of Ede1 and show that they are essential for the normal function of Ede1 during endocytosis. Our findings suggest that Ede1 has a ability form molecular condensates, which promote the initiation and maturation of endocytic sites. The Ede1 condensates exhibit many of the hallmark properties of phase separated liquids.

Results

Ede1 can form dynamic protein condensates

In normal yeast cells, fluorescently tagged Ede1 localizes to endocytic sites at the plasma membrane (Kukulski et al., 2012). However, we discovered previously that under certain experimental conditions Ede1 can also assemble into large condensates (Boeke et al., 2014). These condensates were seen in cells that either overexpressed Ede1, or expressed Ede1 at normal levels, but lacked three early endocytic adaptors. Although these condensates are abnormal structures that have not been observed in wild-type cells, we reasoned that studying them in more detail might provide insights into the mechanism by which Ede1 promotes the assembly of the early endocytic proteins.

To visualize the condensates, we expressed Ede1 fused to enhanced green fluorescent protein (EGFP) from its endogenous locus in haploid wild-type cells, or in cells from which three endocytosis-related genes were deleted (yap1801Δ yap1802Δ apl3Δ, called 3×ΔEA for short). These genes code for early-arriving endocytic adaptor proteins Yap1801, Yap1802, and the α-subunit of the AP-2 complex. Alternatively, we overexpressed EGFP-Ede1 from its endogenous locus under the control of a strong heterologous promoter.

We observed that part of the cellular Ede1-EGFP in the mutant strains localized into condensates that were much brighter than the normal endocytic sites (Figure 1A). The condensates in the overexpression strain were larger and brighter than those in the 3×ΔEA cells (Figure 1B). The condensates usually associated with the plasma membrane, but were also observed away from it (Figure 1A). In contrast, normal endocytic sites are always associated with the plasma membrane. The condensates in the Ede1 overexpression strain were often large enough that their shape was resolvable (Figure 1B). They appeared circular in surface view, and as dome-like structures limited by the plasma membrane in the side view. The Ede1 condensates were remarkably long lived and we have observed individual condensates for up to 1 hour (Figure 1—video 1). This stands in contrast with normal endocytic sites, where Ede1-EGFP typically persists for 1-2 minutes (Stimpson et al., 2009). Despite their stability, some of the condensates appeared to undergo dynamic fission and fusion events, suggesting that they are not solid aggregates (Figure 1C).

Figure 1 with 2 supplements see all
Excess cytosolic Ede1 assembles into condensates in vivo.

(A) Representative images of yeast cells expressing Ede1-EGFP in wild-type (wt) and 3×ΔEA genetic backgrounds, or overexpressing EGFP-Ede1 under the control of the ADH1 promoter. Mutant cell micrographs are shown using the same display range as the wt (top) or their full display range (bottom). Two cells are shown for 3×ΔEA background to display the membrane-associated and cytoplasmic localizations of Ede1 condensates. Scale bars: 2 μm. (B) Representative images of Ede1-EGFP at endocytic sites in wt background, Ede1-EGFP condensates in 3×ΔEA cells, and EGFP-Ede1 overexpression-induced condensates (OE). OE condensates are shown in two different orientations. Each frame is 1.5 μm × 1.5 μm; dotted white line represents the approximate position of the plasma membrane. (C) Two time series of Ede1-EGFP condensates undergoing apparent fusion (top) and fission (bottom) events. Scale bar: 1 μm. (D) Representative images of diploid cells homozygous for the 3×ΔEA background, each expressing Ede1-EGFP and differing in the second Ede1 locus: EDE1-EGFP, EDE1, or ede1Δ. Scale bars: 2 μm. (E) The fraction of cells containing condensates in each strain from panel D. Bars and whiskers show mean ± SD of three independent experiments. A range of 40–70 cells were analyzed per data point.

All three adaptors absent from 3×ΔEA cells interact with Ede1, as well as membrane lipids and protein cargo. As Ede1 does not have known membrane-binding activity, it is likely that the cytosolic pool of Ede1 is increased both in the overexpression and the 3×ΔEA backgrounds, and the excess protein assembles into the condensates. To test whether the formation of condensates in 3×ΔEA background depends on Ede1 concentration, we generated diploid cells homozygous for the three adaptor deletions. We expressed Ede1-EGFP in these cells either from both EDE1 alleles (EDE1-EGFP/EDE1-EGFP), or from one allele in combination with untagged EDE1 (EDE1-EGFP/EDE1) or a deletion of EDE1 (EDE1-EGFP/ede1Δ). The condensates formed in both strains expressing two alleles of EDE1, but not in the strain where only one EDE1 allele was present (Figure 1D). This result suggests that the condensate assembly depends on Ede1 concentration.

The EGFP tag used in these experiments has a weak tendency to dimerize and can induce protein clustering in some in vivo contexts (Costantini et al., 2012). We expressed Ede1 tagged with monomeric fluorescent proteins mNeonGreen, msGFP2, and mCherry in 3×ΔEA cells and observed the same phenotype as with EGFP-tagged Ede1 (Figure 1—figure supplement 1). The observed phenotype is therefore unlikely to have been caused by the choice of the fluorescent tag.

It must also be noted that there is a large difference in brightness between genuine endocytic sites and condensates in mutant cells. Therefore, we chose to saturate the display of those images which show both classes of objects simultaneously, in order for the cells and endocytic sites to remain visible. This difference is demonstrated in panel A of Figure 1.

Ede1 condensates exhibit liquid-like properties

Because of their spherical shapes, concentration dependence, and dynamic behaviors, we hypothesized that the Ede1 condensates might be phase-separated liquid droplets. To test this idea, we first performed fluorescence recovery after photobleaching (FRAP) experiments on Ede1-EGFP condensates in the 3×ΔEA background. After photobleaching, the condensates rapidly recovered most of their fluorescence (Figure 2A). The recovery half-time of a single-exponential FRAP model fitted to an average of 36 events was 22 s, and the mobile fraction was 63%.

Figure 2 with 1 supplement see all
Ede1 structures exchange molecules with the cytoplasm and respond to temperature changes.

(A, B) Fluorescence recovery after photobleaching (FRAP) of Ede1-EGFP condensates in 3×ΔEA cells (A) and endocytic sites in normal cells (B). Plots show mean fluorescence recovery ± SD; n = 36 across four independent experiments (panel A) and n = 14 across three independent experiments (panel B). Representative time series are shown below each plot. Each frame is 1 μm × 1 μm. (C) Time series of a partial bleaching of a condensate in a cell overexpressing EGFP-Ede1. A perceptually uniform color lookup table has been applied to highlight the changes in intensity. Each frame is 1.5 μm × 1.5 μm.(D) Average fluorescence recovery (n = 14) after partial bleaching of condensates in cells overexpressing EGFP-Ede1, as in panel (C). (E) Representative cells after 5-min treatment with indicated concentrations of 1,6-hexanediol. Maximum Z-projections. (F) Ede1-EGFP was imaged in 3×ΔEA cells at different temperatures. Cells were grown and imaged at 24°C. The temperature was raised to 37 and 42°C and returned to 24°C for the indicated amounts of time. Maximum Z-projections. All scale bars: 2 μm.

We then examined the recovery of Ede1 in normal endocytic sites, photobleaching them during total internal reflection fluorescence (TIRF) imaging. We found that Ede1-EGFP also turns over fast at endocytic sites (half-time of 7.8 s and mobile fraction of 91%). The turnover at the endocytic sites was faster and the mobile fraction higher than in the condensates.

We also performed experiments where we bleached the Ede1 condensates only partially, in order to visualize diffusion of fluorescent molecules within the condensates. For these experiments we used cells overexpressing Ede1-EGFP, in which the condensates are larger than in the 3×ΔEA cells. When a subregion of an endocytic condensate was bleached, the fluorescence in the bleached and unbleached regions equalized within several seconds (Figure 2C and D; Figure 2—video 1).

The formation of phase-separated condensates depends on protein concentration and can be affected by environmental factors such as temperature (Molliex et al., 2015; Nott et al., 2015; Franzmann et al., 2018). When the 3×ΔEA cells cultured at 24°C were incubated at 37°C for 5 min, the condensates dissolved while the endocytic sites persisted (Figure 2D). When the temperature was raised to 42°C, Ede1 signal became entirely diffuse. This effect was reversible for both the endocytic sites, which reformed after several minutes, and the condensates, which reappeared within 30 min after return to 24°C.

1,6-Hexanediol is an aliphatic alcohol that disrupts weak protein-protein interactions (Patel et al., 2007) and is used to distinguish between solid and liquid protein aggregates (Kroschwald et al., 2015). Both Ede1 condensates and endocytic patches rapidly disappeared in 3×ΔEA cells upon 1,6-hexanediol treatment (Figure 2E). Interestingly, endocytic patches only fully dissolved at higher 1,6-hexanediol concentrations than Ede1 condensates.

Taken together our results show that Ede1 both in the condensates and at the endocytic sites behaves in a highly dynamic, liquid-like manner.

Next, we sought to determine the concentration of Ede1 in the 3×ΔEA and Ede1 overexpression cells relative to the wild-type cells. We used spinning-disk confocal microscopy to limit the influence of out-of-focus condensates on the quantification of cytosolic intensity. We quantified the mean pixel intensity in entire cell volumes and in small cytosolic regions of cell cross-sections (Figure 3A). The total cellular intensity was on average 122 and 321% of the wild-type in 3×ΔEA and overexpression cells, respectively. The cytosolic intensity was nevertheless uniform across all three strains and we could detect no statistically significant differences (p=0.45 in F-test).

Figure 3 with 1 supplement see all
Condensate formation limits the concentration of Ede1 in the cytoplasm.

(A) Fluorescence intensity was measured in wild-type (wt) and 3×ΔEA cells expressing Ede1-EGFP, or cells overexpressing (oe) EGFP-Ede1 from the ADH1 promoter. Gray points represent mean pixel intensities of entire cell volumes (Cellular), or small regions manually selected from cell cross-sections (Cytosolic). Large points: mean values from independent replicates; central line and whiskers: mean ± SD of replicate means. Pairwise comparisons based on a linear mixed model (n.s., not significant; ***, p<0.001). (B) Two yeast strains were imaged for 8 hr after change of carbon source from glucose to galactose (see Materials and methods). Cells express EGFP-Ede1 under the control of GALS promoter (green channel only), or Ede1-EGFP and Sla1-mCherry expressed from the endogenous loci (green and magenta, respectively). Scale bars: 2 μm. (C) Fluorescence intensity during the expression induction was measured in regions representing entire cells (2 and 4 in panel B) and their cytoplasm (1 and 3). Mean intensity is shown for endogenously expressed (wt) or overexpressed (oe) Ede1 after background subtraction, ±2 × SEM (n = 40 cells for each strain).

We also induced overexpression of EGFP-tagged Ede1 using the weakened galactokinase promoter GALS (Mumberg et al., 1994). We followed the changes in fluorescence intensity over 8 hr after switching carbon source from glucose to galactose (Figure 3B and C; Figure 3—video 1). The cells, in which overexpression was induced after glucose repression, initially showed no Ede1 sites at the membrane. After several hours, Ede1 appeared as transient endocytic sites, and later formed large and stable condensates. The intensity of the cytosolic regions in the overexpression cells never surpassed the cytosolic intensity of wild-type cells, even as the total intensity of the mutant reached approximately 150% of wild-type intensity by the end of the experiment.

These experiments suggest that the cytosolic concentration of Ede1 is buffered by the formation of the condensates. Moreover, the cytoplasmic fluorescence intensity in wild-type cells is already at the limit observed during overexpression. This signifies that the total concentration in wild-type cells is above the critical concentration required for phase separation.

Ede1 condensates recruit other endocytic proteins

We then imaged double-tagged strains to test whether other endocytic proteins colocalize with Ede1 condensates in the 3×ΔEA background (Figure 4A and B). We found that the condensates contain multiple early (Syp1, Ent1, Sla2) and late (End3, Pan1, Sla1) coat proteins, as well as the actin nucleation-promoting factor Las17, known to physically interact with the End3/Pan1/Sla1 complex (Sun et al., 2015; Feliciano and Di Pietro, 2012).

Figure 4 with 2 supplements see all
Endocytic condensates recruit many proteins.

(A) Images of representative cells expressing Ede1-EGFP and indicated endocytic proteins tagged with mCherry in 3×ΔEA background. (B) Fraction of Ede1-EGFP condensates that colocalized with mCherry puncta in each strain from panel A. Bars and whiskers show mean ± SD of three independent experiments. A range of 36–98 cells were analyzed per data point. (C) Montage from timelapse imaging of Ede1-EGFP and Abp1-mCherry during apparent fission of an Ede1 condensate.

Three proteins—Myo5, Abp1, and Rvs167—whose arrival overlaps with actin polymerization at the endocytic sites (Sun et al., 2006) localized to a minority of the condensates (42, 20, and 10%, respectively). We further examined the interaction of condensates and Abp1 using timelapse imaging and found that Abp1-mCherry patches assembled on the condensates transiently (Figure 4C), which explains the partial colocalization. Abp1-mCherry was also present whenever the apparent fission of condensates occurred (Figure 4C, Figure 4—video 1). The appearance of Abp1 during condensate fission suggests that fission could be caused by a force exerted by actin filaments. Some of the proteins we see in the condensates, such as Las17, could potentially trigger actin polymerization.

Costaining with FM4-64 showed that endocytic protein condensates do not contain a significant membrane fraction (Figure 4—figure supplement 1).

Overall, the colocalization experiments showed that the endocytic condensates are complex, and that the proteins contained within them are at least partially functional as they can recruit their interaction partners and are associated with cycles of assembly and disassembly of actin.

Ede1 central region is necessary and sufficient for phase separation

Ede1 is a 1381 amino acid long, multidomain protein (Figure 5A). Its N-terminal region contains three Eps15-homology (EH) domains that interact with asparagine-proline-phenylalanine motifs found on endocytic adaptors such as Ent1/2, Yap1801/2, and Sla2 (Maldonado-Báez et al., 2008). Such repeats of domains interacting with linear motifs are known to promote liquid-liquid phase separation (Li and Elledge, 2012; Banjade and Rosen, 2014). The EH domains are followed by a proline-rich region and a coiled-coil domain (Reider et al., 2009; Lu and Drubin, 2017). The C-terminal half of Ede1 contains a Syp1-interacting region (Reider et al., 2009) and a ubiquitin-associated (UBA) domain.

Figure 5 with 1 supplement see all
The central region of Ede1 is necessary and sufficient for condensate formation.

(A) The domain structure of Ede1 and prediction of disordered and prion-like regions. EH, Eps15-homology domain; UBA, ubiquitin-associated domain. Domains are drawn to scale according to UniProt entry P34216, and numbers above mark domain boundaries used in our constructs. The top plot represents IUPred2a disorder prediction score, with the shaded areas predicted to be disordered by MobiDB-lite consensus method. Prion-like domain (PLD) prediction score was calculated using the PLAAC software. (B) Representative cells expressing full-length (FL) Ede1 and its truncation mutants in wild-type and 3×ΔEA backgrounds. All constructs are C-terminally tagged with EGFP. Maximum intensity projections of 3D volumes, scale bars: 2 μm. (C) The fraction of cells containing condensates in each strain from panel B. Bars and whiskers show mean ± SD of three independent experiments. A range of 37–78 cells were analyzed per data point.

We noticed that the proline-rich region contains a high number of asparagine and glutamine residues, a hallmark of prion-like domains (PLDs) which are proposed to regulate phase separation of many proteins (Alberti et al., 2009; Franzmann et al., 2018; Franzmann and Alberti, 2019). We used PLAAC (Lancaster et al., 2014), a web-based version of the algorithm used by Alberti et al., 2009 to detect prion candidates in yeast proteome, to analyze the Ede1 sequence (Figure 5A). The algorithm detected a 99-amino acid-long prion-like sequence between amino acids 374 and 472, suggesting that this region could also be involved in the phase separation of endocytic proteins. We also consulted IUPred2a (Mészáros et al., 2018) and MobiDB-lite (Necci et al., 2017) algorithms to predict intrinsically disordered regions (IDRs) in Ede1. About 36% of Ede1 is predicted to be disordered; the unstructured regions are contained within the proline- and glutamine-rich region, and between the coiled-coil and the UBA domain.

To understand which features of Ede1 mediate phase-separation, we expressed a series of truncations of Ede1 in both wild-type and 3×ΔEA backgrounds, and analyzed their localization to condensates and endocytic sites (Figure 5B and C). In our 3×ΔEA background, the N- and C-terminal regions were dispensable for condensate formation. Surprisingly, the Ede1 central region consisting of amino acids 366–900 (the unstructured PQ-rich region and the coiled-coil domain) localized to large condensates in both the 3×ΔEA and wild-type backgrounds. It was also the minimal construct to form these condensates, as constructs containing only the coiled-coil or the PQ-rich region showed diffuse cytoplasmic localization.

For the wild-type background, our results are largely consistent with previously published results about the localization of Ede1 mutants (Boeke et al., 2014; Lu and Drubin, 2017). Namely, the coiled-coil domain was necessary for Ede1 to assemble into endocytic sites, while the N- and C-terminal parts of Ede1 were individually dispensable.

We analyzed the concentration of truncated variants in both backgrounds by quantitative western blotting (Figure 5—figure supplement 1). The truncated Ede1 variants were expressed at higher levels than the full-length protein. This observation suggests that the loss of condensates in strains with truncated Ede1 is indeed caused by missing motifs rather than lowered concentration. We also confirmed that the concentration of full-length Ede1 increased in the 3×ΔEA background. It is unclear whether this represents a compensatory genetic mechanism, or if condensation is initially caused by reduced plasma membrane recruitment and subsequently interferes with protein degradation.

The functional significance of the Ede1 central region

To test the role of the central region of Ede1 in endocytosis, we created EGFP-tagged Ede1 mutants with internal deletions of amino acids 366–590 (Ede1ΔPQ), 591–900 (Ede1ΔCC), and 366–900 (Ede1ΔPQCC). Ede1ΔPQCC failed to localize to endocytic sites, whereas the two single-domain Ede1ΔPQ and Ede1ΔCC deletion mutants were still punctate, but more diffuse compared to full-length Ede1-EGFP (Figure 6A).

Figure 6 with 1 supplement see all
Ede1 features necessary for phase separation are also crucial for its function.

(A) Representative cells expressing full-length Ede1 and three internal Ede1 deletion mutants: Ede1ΔPQ (Δ366-590), Ede1ΔCC (Δ591-900), and Ede1ΔPQCC (Δ366-900) tagged with EGFP. (B) Representative cells expressing Sla1-EGFP and indicated Ede1 mutants. (C, D) Sla1 patch density and lifetime in Ede1 mutants. Large points represent mean measurements from independently repeated datasets. Central line and whiskers denote the mean ± SD calculated from dataset averages. Gray points show individual observations. Letters denote pairwise comparisons based on Tukey-Kramer test; groups which do not share any letters are significantly different at α = 0.05. Scale bars: 2 μm.

Figure 6—source data 1

Source data, code, and statistical details (panel C).

https://cdn.elifesciences.org/articles/72865/elife-72865-fig6-data1-v2.zip
Figure 6—source data 2

Source data, code, and statistical details (panel D).

https://cdn.elifesciences.org/articles/72865/elife-72865-fig6-data2-v2.zip

Next, we tested if these Ede1 mutants had endocytic defects by using Sla1 as a reporter of the late phase of endocytosis. We tagged Sla1 with EGFP in Ede1 mutant strains (Figure 6B) and quantified the density and lifetimes of endocytic sites (Figure 6C and D). In ede1Δ and ede1ΔPQCC cells, the mean number of endocytic events marked by Sla1-EGFP per μm2 was reduced by 46 and 43% of the wild-type, respectively. Consistent with their effects on Ede1 recruitment, the ede1ΔPQ and ede1ΔCC mutations caused intermediate reduction in patch density (by 24 and 26%, respectively). All differences from the wild type were statistically significant (p<0.001 in Tukey-Kramer test). The difference between ede1Δ and ede1ΔPQCC was not statistically significant (p=0.86).

The Sla1 lifetimes were likewise affected by the deletion of Ede1 central region. In ede1Δ, Sla1-EGFP lifetime was decreased by 29% and in ede1ΔPQCC, by 28%. The deletions of individual regions again showed intermediate defects (13 and 18% for Ede1ΔPQ and Ede1ΔCC, respectively). All differences from the wild type were statistically significant (p<0.01 in Tukey-Kramer test). The deletion of the entire EDE1 gene was not significantly different from the ede1ΔPQCC mutant (p = 0.99).

The Ede1ΔPQCC mutant does not localize to endocytic sites. We wanted to test if the N- and C-terminal domains alone could support endocytosis if a strong interaction with another endocytic protein was introduced. We generated cells coexpressing Ede1ΔPQCC-mCherry-FKBP and Syp1-FRB or Sla2-FRB in order to link the Ede1 mutant to another component of the early coat via the rapamycin-inducible FKBP-FRB dimerization system (Haruki et al., 2008). Recruitment to Syp1 caused the Ede1ΔPQCC-mCherry-FKBP signal to become more prominent around the bud necks, but did not rescue membrane patch formation, whereas recruitment to Sla2 partially rescued the patch localization of Ede1ΔPQCC (Figure 6—figure supplement 1). The average Sla1 density did not significantly change in either of these yeast strains upon rapamycin treatment (p=0.53 and p=0.59 between treated and untreated cells in Syp1-FRB and Sla2-FRB strains, respectively).

Taken together, our results show that the central region is essential for Ede1 to promote efficient endocytosis and to regulate the timing of coat maturation.

In ede1Δ cells, many of the early endocytic proteins fail to localize to endocytic sites (Stimpson et al., 2009; Carroll et al., 2012). We therefore visualized the localization of early proteins in Ede1 mutants lacking the central region.

Different proteins were affected by the Ede1 central deletions in different ways (Figure 7), consistent with the work done on ede1Δ mutants. The localization of Apl1 (β-subunit of the AP-2 complex) was the most severely disrupted. Apl1-EGFP patches were less defined in Ede1ΔPQ background, and undetectable in Ede1ΔCC or Ede1ΔPQCC cells. In these cells, Apl1-EGFP signal was dispersed in the cytoplasm, with a faint presence around the bud neck. Syp1 and Yap1801 remained localized to the membrane in all of the mutants, but the signal became more diffuse along the membrane and the patches less defined. This effect was the strongest for Ede1ΔPQCC, with intermediate effects in Ede1ΔPQ and Ede1ΔCC cells. However, Ent1 and Sla2 still assembled into endocytic patches in all of the Ede1 mutants. Taken together, our results indicate that the Ede1 central region is essential to concentrate early endocytic proteins.

Ede1 central deletion mutants are defective in early protein localization.

Maximum-intensity projections of 3D volumes are shown for representative cells with different early proteins tagged with EGFP. The strains express Ede1 species indicated at the top. Scale bars: 2 μm.

Ede1 central region can be replaced by other prion-like domains

We wanted to test whether the loss of function in ede1ΔPQCC cells in respect to Sla1 density could be rescued by heterologous protein sequences, such as IDRs, globular domains, or coiled-coils (Figure 8A).

Figure 8 with 1 supplement see all
Prion-like domains can partially replace Ede1 central region.

(A) Domain structure of the Ede1 central region replacement constructs. Amino acids 366–900 of Ede1 were replaced with prion-like intrinsically disordered region (IDR) sequences, monomeric (mCherry) or dimeric (dTomato) fluorescent proteins, and dimeric (Khc) or tetrameric (Eg5) coiled-coils. All mutants were expressed from the Ede1 locus under the control of the native promoter. (B) Representative cells expressing indicated Ede1 mutants tagged C-terminally with msGFP2. (C) Quantification of Sla1-EGFP patch density in strains expressing indicated Ede1 mutants. Large points represent mean measurements from independently repeated datasets. Central line and whiskers denote the mean ± SD calculated from dataset averages. Gray points show individual cells from all datasets. At α = 0.05, all mutants are significantly different from wild type, and groups marked with an asterisk (*) are significantly different from Ede1ΔPQCC (Tukey-Kramer test; a complete table of pairwise comparisons and effect sizes can be found in Figure 8—source data 1). All scale bars: 2 μm.

Figure 8—source data 1

Source data, code, and statistical details (panel C).

https://cdn.elifesciences.org/articles/72865/elife-72865-fig8-data1-v2.zip

First, we replaced amino acids 366–900 of Ede1 with different IDRs. We considered three factors for choosing the replacement IDRs: known phase separation activity, Q/N content, and similarity to the Ede1 IDR sequence. We chose the region of Sup35 spanning the N and M regions based on its known phase separation activity in yeast cells (Franzmann et al., 2018). We chose the IDR of Snf5 because of its high score in the prion-like screen by Alberti et al., 2009, reflecting a Gln-rich amino acid sequence, and the IDR of Whi3, because the method developed by Zarin et al., 2019 indicated it as one of the sequences most similar to the PQ-rich region of Ede1. Finally, we also included the low-complexity domain of human FUS for its well-known tendency to phase separate and form hydrogels (Patel et al., 2015; Kato et al., 2012).

We also replaced the central region of Ede1 with several structured domains. We used the red fluorophores mCherry and dTomato as globular linkers assuming respectively monomeric and dimeric states. We also used the coiled-coil domains from two kinesin motors as oligomeric rod-shaped linkers: the kinesin-1 heavy chain (Khc amino acids 335–931) from Drosophila melanogaster and the human kinesin-5 Eg5 (amino acids 358–797), which form dimers and tetramers in their respective contexts (de Cuevas et al., 1992; Scholey et al., 2014).

All of the Ede1 central region replacement constructs were expressed from the endogenous genomic locus under the control of the native promoter.

We assessed the localization of Ede1 constructs tagged with msGFP2 (Figure 8B, Figure 8—figure supplement 1). We found that the localization was partially rescued by the insertion of Whi3, Snf5, and Sup35 IDRs, as well as the oligomeric linkers (dTomato, Khc, Eg5), in place of the deleted central region. The localization was not rescued by the FUS low-complexity region or the monomeric globular linker mCherry.

We also assessed the density of Sla1-EGFP patches in cells expressing untagged Ede1mCherry, Ede1dTomato, Ede1Khc, Ede1Eg5, Ede1Snf5, or Ede1Sup35 (Figure 8C). All differences from wild type were statistically significant (p<0.001). No single replacement mutant was able to fully rescue the Sla1 patch density defect present in ede1ΔPQCC cells. Only ede1Sup35 was able to significantly rescue the Sla1 density in respect to the deletion of Ede1 central region. In ede1ΔPQCC and ede1Δ cells, the mean density of Sla1 patches was reduced by 40 and 48%, respectively. In ede1Sup35, the mean density was only reduced by 25% from wild type, respectively.

These results support the hypothesis that prion-like domains can aid clustering of the endocytic proteins.

Ede1 central region can cluster a heterologous lipid-binding protein

We hypothesized that the phase separation mediated by Ede1 central region is able to cluster membrane-associated proteins. To test our hypothesis, we fused the Ede1 central region to a diffusely membrane-bound protein. We created a GFP-Ede1366-900-2×PH(PLCδ) construct, based on a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) probe developed by Stefan et al., 2002, by inserting the Ede1 central region between GFP and a tandem repeat of pleckstrin homology (PH) domain of phospholipase C δ1 (PLCδ). The original GFP-2×PH(PLCδ) construct is distributed homogeneously on the plasma membrane, while GFP-Ede1366-900 alone localized to bright intracellular condensates. In contrast, the fusion construct localized to the plasma membrane, forming puncta that resembled endocytic sites (Figure 9A,B).

Figure 9 with 3 supplements see all
Fusion with Ede1 central region changes the distribution of a PI(4,5)P2 probe.

(A) Maximum projections of cells expressing GFP-2 × PH and GFP-Ede1366-900 from a yeast centromeric plasmid under the control of TDH3 promoter. White dotted line shows cell outline. (B) Cells expressing GFP-Ede1366-900-2×PH from a multicopy plasmid. Examples of different structures classified as ‘Diffuse’, ‘Patches’, or ‘Networks’. (C) and (D) The same construct was expressed from four centromeric plasmids under different promoters. Individual cells were classified as in panel B. Plots show percentage of cells falling into each class per promoter (C), and mean cell pixel intensity per class (D). (E) Movies of cells coexpressing GFP-Ede1366-900-2×PH and Sla1-mCherry were acquired using TIRF microscopy. Single frame from a representative movie; points labeled ‘1’ and ‘2’ mark the top and bottom of the kymograph (F). Scale bars: 2 μm. All cells in this figure: SLA1-mCherry::KANMX4, ede1Δ::natNT2.

We also noticed subpopulations of cells with different localization patterns of the construct (Figure 9B). We speculated that the variable patterns were caused by heterogeneity in protein expression level due to plasmid copy number variation. To test that hypothesis, we expressed GFP-Ede1366-900-2×PH(PLCδ) from centromeric plasmids containing four different promoters of increasing strength (Mumberg et al., 1995). We classified the localization of the construct in these cells as ‘diffuse’, ‘punctate’, or ‘networked’. We found that the tendency to cluster into different patterns correlated with promoter strength and the expression level. Low expressing cells had more diffuse localization of the construct, and separated into puncta or well-separated regions as the concentration increased (Figure 9C,D).

The puncta formed by the GFP-Ede1366-900-2×PH(PLCδ) construct were stable over long imaging periods, but dynamically recruited the late coat protein Sla1 (Figure 9F, Figure 9—video 1, Figure 9—video 2). Sla1-mCherry persisted at these sites with similar lifetimes as during normal endocytosis indicating that the condensates can recruit endocytic coat components. Unlike full-length Ede1 at the endocytic sites, the chimeric construct does not undergo cycles of assembly and disassembly. When we photobleached the structures formed by highly expressed GFP-Ede1366-900-2×PH(PLCδ) we saw no recovery (Figure 9—figure supplement 1A), indicating that without the terminal domains, Ede1 central region might form solid, rather than liquid-like, structures. 10% 1,6-hexanediol also failed to dissolve the GFP-Ede1366-900-2×PH(PLCδ) structures (Figure 9—figure supplement 1B). Structures formed by strongly overexpressed GFP-Ede1366-900 were partially dissolved by 10% 1,6-hexanediol. This suggests that the stability of GFP-Ede1366-900-2×PH(PLCδ) might also be modulated by the PH domains interacting with the membrane. In addition, we noticed that 1,6-hexanediol treatment caused membrane deformations, or possible clustering of the PH domains expressed without the Ede1 fusion (Figure 9—figure supplement 1B). This observation is consistent with the previous reports of wide-ranging effects of 1,6-hexanediol (Kroschwald et al., 2015), and underscores that 1,6-hexanediol experiments need to be interpreted with caution.

These results show that directing the Ede1 central region to the plasma membrane is sufficient to create puncta on the membrane in a concentration-dependent manner. Surprisingly, these long-lived sites can repeatedly recruit endocytic coat components.

Discussion

Ede1 is the key organizer of the early phase of endocytosis in yeast (Stimpson et al., 2009; Boeke et al., 2014; Lu and Drubin, 2017). Our results indicate that the large clusters of Ede1 observed previously in mutant cells (Boeke et al., 2014) are in fact phase-separated protein droplets. Moreover, we found that the cytosolic concentration of Ede1 in normal cells is at the same critical limit as in the mutant cells harboring Ede1 droplets. This suggests that liquid phase separation might be the mechanism through which Ede1 concentrates proteins at the early endocytic sites. We identified the central region of Ede1— containing a coiled-coil and a prion-like domain— as necessary for both the condensate formation, and for Ede1 to promote the initiation of endocytosis. We also found that heterologous prion-like domains can partially replace the Ede1 central region in endocytosis. We also demonstrated that the central region of Ede1 fused to a lipid-binding domain can condense on the plasma membrane. These findings suggest a potential link between endocytic assembly and the phenomenon of protein phase separation and raise questions about the material properties of the endocytic sites at different stages. They also highlight a possible novel role for disordered, prion-like regions found in numerous endocytic proteins (Malinovska et al., 2013).

Ede1 forms liquid protein droplets

Ede1 forms large condensates under conditions where the stoichiometry between Ede1 and the endocytic adaptor proteins is altered, such as overexpression of Ede1 or deletion of three early adaptors (Boeke et al., 2014, Figure 1).

We showed that these condensates are liquid, phase-separated droplets according to the following criteria: (a) observation of liquid-like behaviors, (b) molecule turnover, (c) dependence on a critical component concentration, (d) dependence on temperature, and (e) susceptibility to dissolution by 1,6-hexanediol.

We show that the Ede1 condensates undergo apparent fusion and fission events, the latter caused possibly by polymerizing actin filaments (Figure 4C, Figure 4—video 1). The Ede1 molecules exchange between the condensates and the cytosolic pool (Figure 2A) and, importantly, Ede1 molecules also rapidly diffuse within the condensates (Figure 2C and D). The formation of the Ede1 condensates depends on the cellular concentration of Ede1 (Figures 1D and 3). The condensates dissolve rapidly and reversibly in response to temperature changes (Figure 2F), and are sensitive to treatment with 1,6-hexanediol (Figure 2E). In agreement with our findings in yeast, Day et al., 2021 have shown that Eps15, the mammalian homolog of Ede1, can phase separate in vitro.

The Ede1 condensates are clearly distinct from endocytic sites by the virtue of size, brightness, and long-term stability. We assign them no function, other than as a tool used to study the properties of Ede1. However, Wilfling et al., 2020 studied Ede1 condensates in the 3×ΔEA and Ede1 overexpression strains in parallel to our work. They describe a selective autophagy pathway mediated by Ede1, and propose that autophagy of phase-separated condensates might be a major route through which cells remove misfolded or unneeded endocytic proteins. The ability of Ede1 to cluster endocytic proteins could therefore play a dual role in endocytosis and autophagy.

Endocytic sites: solid or liquid?

The prevailing model of endocytosis focuses on the growing clathrin lattice as the driver of protein assembly (Kirchhausen et al., 2014; Cocucci et al., 2012). Indeed, clathrin is a major interaction hub in endocytosis, and a scaffold with a well-defined structure. Nevertheless, several lines of evidence suggest that this model of assembly might be incomplete.

In yeast, the early proteins can assemble in the absence of clathrin. In fact, many endocytic sites in clc1Δ cells stall during the early phase, but persist on the plasma membrane (Carroll et al., 2012). On the other hand, in the absence of Ede1, numerous early adaptors do not assemble at all, or assemble only for the duration of the membrane-bending phase (Stimpson et al., 2009; Carroll et al., 2012). These two observations demonstrate that Ede1 can sustain the early sites independently of the clathrin lattice. We hypothesize that Ede1 performs this function by undergoing liquid-liquid phase separation on the plasma membrane.

FRAP shows that numerous proteins involved in endocytosis and the actin cytoskeleton continuously turn over at the endocytic sites (Skruzny et al., 2012; Lacy et al., 2019). Ede1 is one of such proteins (Figure 2B). This observation is consistent with a phase separation mechanism, although it does not prove it. Alternative mechanisms could explain fast turnover, such as ‘treadmilling’ of actin monomers or dynamic binding of adaptors to the clathrin lattice. Even clathrin itself shows fluorescence recovery as individual triskelia are replaced within the scaffold (Wu et al., 2001; Avinoam et al., 2015; Chen et al., 2019).

Several hallmark criteria frequently associated with liquid phase separation can also be explained by other mechanisms of compartmentalization. This fact has become intensely debated in the context of the nucleus, where bridging of multiple DNA sites could create an appearance of phase-separated compartments (McSwiggen et al., 2019; Peng and Weber, 2019). Similarly, weak binding to the clathrin lattice could explain the enrichment of proteins at endocytic sites with high turnover and susceptible to dissolution by temperature and 1,6-hexanediol.

Our observations point to a mixed model, in which the structured lattice exists alongside a liquid phase formed by unstructured interactions. The existence of large Ede1 condensates is in itself one of the predictions generated by a phase separation model. A significant consequence of phase separation is that above a critical value, further increase of total component concentration leads to changes in relative volume, but not concentration, of the dense and the light phases. This is in contrast with the scaffold-binding model, where the size of the assemblies formed by endocytic proteins would be limited by the clathrin lattice. In addition, we have observed buffering of cytoplasmic Ede1 concentration during overexpression and in different genetic backgrounds. The cytoplasmic concentration in wild-type cells reached the limit observed in Ede1 overexpression, suggesting that the normal Ede1 concentration is sufficient for its phase separation on the plasma membrane. The small fraction of wild-type cells which contain larger Ede1 condensates (Figures 1E and 5C, Wilfling et al., 2020) could very well be the consequence of natural variability in expression levels. The concentration buffering also suggests that the phase separation of Ede1 is driven primarily by homotypic interactions (Riback et al., 2020).

The qualitative criteria for liquid phase separation—sensitivity to temperature and 1,6-hexanediol—apply to Ede1 at the endocytic sites as well as to the large endocytic condensates. Curiously, the sites appear more stable against both of these treatments than the large condensates. This could be explained by the fact that adaptor binding confines Ede1 at the endocytic sites to the plane of the membrane. As such, the critical concentration could be lower than for the formation of 3D droplets, as shown previously in vitro for the Nephrin/Nck/N-WASP system (Banjade and Rosen, 2014). A complementary explanation could be that genuine endocytic sites are stabilized by other interactions, such as those within the clathrin lattice, or the lattice formed by Sla2 and Ent1/2 in the presence of PI(4,5)P2.

The small size of the endocytic sites prevents the direct visualization of liquid-like shape changes. Super-resolution imaging of endocytic proteins in fixed cells revealed that Ede1 and Syp1 form larger and more amorphous structures than clathrin and its adaptors (Mund et al., 2018). The super-resolution experiments also suggest that even before it disassembles, Ede1 becomes progressively excluded from the center of the sites. Eps15 and FCHo1 behave in a similar fashion in mammalian cells (Sochacki et al., 2017). As the intermediate and late coat proteins form stable patches with low turnover (Skruzny et al., 2012), we propose that a liquid-like early module is displaced from the center of the invagination by the formation of a solid coat.

Ede1 is one of the most heavily phosphorylated proteins in yeast endocytosis (Lu et al., 2016), and it can be ubiquitylated as well as bind ubiquitin. Phosphorylation can regulate phase separation (Monahan et al., 2017; Larson et al., 2017), and the phosphorylation of Ede1 might regulate its state at the endocytic sites.

Our chimeric construct GFP-Ede1PQCC-2×PH combines a lipid-binding domain with the central region of Ede1. This construct forms bright structures on the surface of the plasma membrane, and the area covered by these structures appears larger in cells with higher expression levels (Figure 9). The puncta formed by GFP-Ede1PQCC-2×PH repeatedly recruit transient assemblies of the late coat protein Sla1. This suggests that the chimeric construct can initiate functional endocytic events. However, the GFP-Ede1PQCC-2×PH puncta persist over long imaging periods and do not disassemble after Sla1 internalization. Structures formed by GFP-Ede1PQCC-2×PH also do not recover fluorescence (Figure 9—figure supplement 1). This suggests that while the central region of Ede1 can drive condensation, the terminal regions are needed to maintain the liquid state.

It must be noted that we do not fully understand the nature of the microdomains formed by the GFP-Ede1PQCC-2×PH construct. For example, all known interaction motifs of Ede1 are located inside the terminal regions. It is thus unclear how the central region could recruit other endocytic proteins.

Prion-like domains are enriched in yeast endocytic proteins (Alberti et al., 2009; Malinovska et al., 2013), many of which contain polyglutamine tracts longer than that of Ede1. These disordered, low-complexity regions had been previously considered mere linkers (Dafforn and Smith, 2004), but we show that the prion-like region of Ede1 is important for its condensation. Our results also show that unrelated prion-like domains can partially replace the function of Ede1 central region, even without the coiled-coil domain (Figure 8). How—or if—the endocytic proteins achieve specificity during recruitment of prion-like domains is an open question. It has also been proposed that phase separation of prion-like domains could provide force for membrane bending (Bergeron-Sandoval et al., 2021).

The coiled-coil domain of Ede1 is also critical for its phase separation, and even more important to the function of Ede1 than the prion-like domain (Figure 6; Lu and Drubin, 2017). The coiled-coil of Eps15 can form dimers and tetramers (Tebar et al., 1997; Cupers et al., 1997), and fluorescence correlation spectroscopy (FCS) data suggests that cytosolic Ede1 can form dimers and higher-order oligomers (Boeke et al., 2014). Multivalency is a critical characteristic of phase-separating proteins (Li et al., 2012; Banani et al., 2016), and oligomerization via the coiled-coil domain could promote phase separation by increasing the valency of other interactions. Less commonly, coiled-coils can also form phase-separating networks in absence of other interaction motifs, such as in the case of centrosome scaffold SPD-5 (Woodruff et al., 2017). In our experiments, replacing the central region of Ede1 with heterologous coiled-coils partially rescued Ede1 localization, but not the late phase defect, of ede1ΔPQCC cells.

Materials and methods

Yeast strains and plasmids

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The list of yeast strains and yeast plasmids used in this study is provided in Supplementary file 1. These materials are available upon request to the corresponding author.

Cells were maintained on rich medium at 24 or 30°C. C-terminally tagged or truncated mutants were generated via homologous recombination with PCR cassettes as described by Janke et al., 2004. N-terminal truncation and internal domain deletion or replacement mutants of Ede1 were generated by first constructing the desired mutant gene in a pET-based plasmid using PCR mutagenesis or ligation-independent cloning (Li et al., 2012). The mutated Ede1 sequence was then amplified by PCR using primers containing 50 bp overlap with 5’ (forward primer) 3’ (reverse primer) UTR sequences of EDE1. The PCR product was transformed into ede1Δ::klURA3 cells. The transformants were selected for on plates containing 5-fluorootic acid, and confirmed by colony PCR and genomic sequencing.

Sequences coding for IDRs replacing Ede1 core region in Figure 8 were obtained from several different sources. The coding sequences of Snf5 and Whi3 IDRs were amplified by PCR from the yeast genome and were confirmed identical to the sequences in the S288C reference genome (SGD:S000000493 and SGD:S000005141, respectively). The coding sequence of amino acids 1–253 of the Sup35NM3 mutant (Franzmann et al., 2018) was cloned from a plasmid kindly provided by Titus Franzmann. The coding sequence of human FUS low-complexity region (amino acids 2–214 from UniProt entry Q6IBQ5) was codon optimized for yeast expression, and synthesized with the rest of the Ede1 sequence by Synbio Technologies (New Jersey). The sequence coding for amino acids 335–931 of D. melanogaster kinesin-1 was cloned from Addgene plasmid K980 (#129762), a gift from William Hancock. The sequence coding for amino acids 358–797 of Homo sapiens kinesin-5 was cloned from Addgene plasmid mCherry-Kinesin11-N-18 (#55067), a gift from Michael Davidson.

Plasmids used in Figure 9 were based on pRS426-GFP-2×PH(PLCδ) (Stefan et al., 2002), a kind gift from Scott Emr. The Ede1366-900 coding sequence was inserted into this plasmid after the last GFP codon using ligation-independent cloning. GFP-2×PH(PLCδ) and GFP-Ede1366-900-2×PH(PLCδ) were then subcloned to a pRS416-based plasmid p416-GPD under the control of TDH3 promoter (Mumberg et al., 1995) using BamHI and SalI restriction sites. GFP-Ede1366-900-2×PH(PLCδ) was also subcloned into p416-CYC1, p416-ADH1, and p416-TEF1 (Mumberg et al., 1995) using the same restriction sites.

The sequence of the msGFP2 fluorophore (Valbuena et al., 2020) was cloned into a PFA6a-based tagging plasmid from Addgene plasmid #135301, a kind gift from Benjamin Glick.

Live cell imaging

Yeast cells were grown to OD600 between 0.3 and 0.8 at 24°C in low-fluorescence synthetic drop-out medium lacking tryptophan, or tryptophan and uracil if required for plasmid maintenance. Cells were attached to cover slips coated with 1 mg ml−1 concanavalin A.

Widefield microscopy

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Widefield micrographs were obtained on an Olympus IX81 widefield microscope equipped with a 100×/NA1.45 objective and an ORCA-ER CCD camera (Hamamatsu), using an X-CITE 120 PC (EXFO) metal halide lamp as the illumination source. The excitation and emission light when imaging EGFP- and mCherry-tagged proteins were filtered through the U-MGFPHQ and U-MRFPHQ filter sets (Olympus). The 3D stacks were acquired with 0.2 μm vertical spacing. The microscope was controlled using the MetaMorph software (Molecular Dynamics).

Total internal reflection fluorescence microscopy

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All TIRF movies were recorded on an Olympus IX83 widefield microscope equipped with a 150×/NA1.45 objective and an ImageEM X2 EM-CCD camera (Hamamatsu) under the control of the VisiView software (Visitron Systems). The 488 nm and 561 nm laser lines were used for illumination of GFP- and mCherry-tagged proteins. Excitation and emission were filtered using a TRF89902 405/488/561/647 nm quad-band filter set (Chroma). Laser angles were controlled by iLas2 (Roper Scientific).

Fluorescence recovery after photobleaching

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Bleaching of Ede1-EGFP in endocytic condensates (Figure 2A and B) was performed using a custom-built set-up that focuses a 488-nm laser beam at the sample plane, on the Olympus IX81 widefield microscope described above. The diameter of the bleach spot was approximately 0.5 μm.

Bleaching of unperturbed endocytic sites (Figure 2C) was performed with a 405 nm laser line controlled by the iLas2 targeting system during simultaneous excitation with 488 nm and 561 nm lasers in TIRF mode on the Olympus IX83 microscope described above. The emission light was collected through a Gemini beam splitter (Hamamatsu) equipped with a Di03-R488/561-t1 dichroic, and FF03-525/50-25 and FF01-630/92-25 emission filters (Semrock).

Spinning disk microscopy

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Spinning disk confocal imaging (Figure 3) was performed in the Photonic Bioimaging Center at the University of Geneva using a Nikon Eclipse Ti1 microscope equipped with a CSU-W1 spinning disk (Yokogawa) using a 100×/NA1.49 objective, an sCMOS Prime 95B camera (Photometrics), and 488 nm and 561 nm lasers as the illumination source.

Induction of protein expression

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For the induction of expression from GALS promoter during live-cell imaging (Figure 3), cells were thawed and grown for several days on Synthetic Complete medium agar plates with 2% galactose as the sole carbon source. The cells were then cultured overnight in a low-fluorescence synthetic drop-out liquid medium with no tryptophan and 2% raffinose as the sole carbon source. The cells were diluted in the morning into the same medium with 2% glucose as the carbon source. The cells were attached to cover slips as described above. Finally, the carbon source in the medium was switched to 2% galactose before the start of the imaging.

Image and data analysis

All code used in this study is available as a single repository at https://github.com/matkozak/KozakAndKaksonen2022, (copy archived at swh:1:rev:5441acf218619f2b03d90633613cccc373c6fe8a; Kozak, 2022). General image analysis was performed using the Fiji distribution of ImageJ (Schindelin et al., 2012; Rueden et al., 2017). All display images were corrected for background fluorescence using the rolling ball algorithm of ImageJ, and movies were corrected for photobleaching using a custo m ImageJ macro. Plots and statistical analyses were generated using R.

FRAP experiments

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FRAP experiments performed on Ede1-EGFP condensates were analyzed according to Phair et al., 2004. Mean fluorescence values were measured from regions of interest representing the background, the cell, and the condensate. A custom-written R script (available in the article repository) was used to subtract background fluorescence, correct for photobleaching and normalize the values between 0 (corrected fluorescence immediately after photobleaching) and 1 (mean corrected fluorescence of 5 s before photobleaching). The recovery curves of individual experiments were aligned to bleach time and averaged. Condensates that showed lateral or axial movement during the acquisition were manually excluded from the averaging. The average was fitted to a single exponential equation from which the mobile fraction and recovery half-time were calculated.

For FRAP experiments performed on native endocytic sites, the background fluorescence was first subtracted from the TIRF images using the ImageJ rolling ball algorithm. EGFP and mCherry fluorescence of single endocytic patches were measured within a circle with a radius of three pixels around the patch centroid position. A custom-written R script was used to calculate the fluorescence recovery much in the same way as for the FRAP of condensates, but no further corrections were made for background signal or imaging-induced photobleaching. To calculate average recovery, we manually selected only events in which Abp1-mCherry signal peaked at least 60 s after bleach time to exclude the effect of Ede1 disassembly at the end of endocytic events.

Patch numbers and lifetimes

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For estimating the number of patches per membrane area, we analyzed single nonbudding cells. The patches were thresholded and counted using a custom Python script available in the article repository. We estimated the cell surface area by measuring the area of the cross-section from maximum intensity projection and multiplying it by 4, under the assumption that an unbudded yeast cell is approximately spherical. For estimating patch lifetimes, we tracked endocytic events using ParticleTracker from the MOSAIC suite (Sbalzarini and Koumoutsakos, 2005) and multiplied trajectory length by the frame rate.

Cytosolic and total cellular intensity

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To obtain cytosolic intensity of Ede1-EGFP, 5 × 5-pixel square regions away from the condensates and vacuoles were manually measured in ImageJ. To measure total cellular intensity, individual cells were cropped in ImageJ. A custom Python script was applied to the cropped cells to generate masks based on Rvs167-mCherry fluorescence, and subsequently measure the Ede1-EGFP signal intensity in the masked region.

Cell classification

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To calculate the percentages of Ede1-EGFP condensates colocalizing with mCherry puncta in Figure 4, cells containing Ede1-EGFP condensates in Figure 5 and cells showing different GFP-EDE1366-900−2×PH localization patterns in Figure 9, single cells were cropped from imaging fields based on a neutral signal (GFP in the case of Figure 4 and brightfield image for Figures 5 and 9). Next, an ImageJ macro was used to display random images from the dataset and the experimenter would assess the presence of the tested phenotype with no knowledge of which strain was being analyzed.

Western blotting

A 300 μl of ice-cold trichloroacetic acid was added to 5 ml of exponentially growing yeast cultures. The cells were pelleted by centrifugation, washed with cold acetone, and dried in a vacuum concentrator. The pellets were resuspended in 100 μl of urea buffer (25 mM Tris-HCl pH 6.8, 6 M urea, 1% SDS) and homogenized by shaking with 200 μl of glass beads. The samples were heated at 95°C for 5 min, mixed with 100 μl 2× SDS loading buffer and centrifuged at 16, 000× g for 5 min.

The samples were subjected to electrophoresis on 4–20% Precast Protein Gels (Bio-Rad) and transferred onto a nitrocellulose membrane using an iBlot2 device (ThermoFischer Scientific). The membranes were blocked for 30 min with 5% bovine serum albumin in PBS-Tween and incubated with primary antibodies overnight at 4°C. The membranes were washed in PBS-Tween, incubated with fluorescent secondary antibodies for 1 hr and washed in PBS-Tween. The fluorescence was measured on an Odyssey scanner (LI-COR Biosciences).

Antibodies

Ede1 constructs were detected using an anti-GFP mouse monoclonal antibody (ab291, Abcam) at 1/2000 dilution, and an anti-Hog1 rabbit polyclonal antibody (sc-9079, Santa Cruz Biotechnology) at 1/1000 dilution was used as a loading control. Donkey antimouse IRDye 680 and anti-rabbit IRDye 800 secondary antibodies (926–68072 and 926–32213 respectively, LI-COR Biosciences) were used at a 1/10,000 dilution.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting source data files.

References

  1. Book
    1. Li MZ
    2. Elledge SJ
    (2012) SLIC: A method for sequence- and ligation-independent cloning
    In: Peccoud J, editors. Gene Synthesis: Methods and Protocols, Methods in Molecular Biology. Totowa, NJ: Humana Press. pp. 51–59.
    https://doi.org/10.1007/978-1-61779-564-0_5

Decision letter

  1. María Isabel Geli
    Reviewing Editor; Institut de Biología Molecular de Barcelona (IBMB), Spain
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands
  3. Stephen J Royle
    Reviewer; Warwick Medical School, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your paper entitled "Phase separation of Ede1 promotes the initiation of endocytic events" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Maria Isabel Geli as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as senior editor. The following individual involved in review of your submission has agreed to reveal their identity: Stephen J Royle (Reviewer #2).

We are sorry to inform you that at this point the manuscript cannot be considered for publication in eLife.

In summary, there is a consensus on the importance of the scientific question addressed by your work, and on the nature of the Ede1 condensates when it is overexpressed or in the endocytic adaptor mutant background. However, 2 of 3 reviewers considered that the manuscript does not demonstrates that Ede1 really undergoes liquid-liquid phase separation at endocytic sites under physiological conditions and whether this property is required for its endocytic function. The concerns follow.

1. Several of the key experiments aimed at showing phase separation yield different results for the condensates and the endocytic sites. Basically, they disassemble at different temperatures and at different hexanediol concentrations. Together with the fact that FRAP per se does not demonstrate that Ede1 forms a separate liquid phase at endocytic sites, the data rather suggest that the Ede1 condensates and the Ede1 endocytic patches have different properties.

2. The experiment showing that domains required to form the condensates are required for endocytosis is not really conclusive because, among other considerations, there seems to be an inverse correlation between the capacity of the different mutants to be recruited at endocytic sites and their capacity to sustain endocytosis. Under these circumstances, the endocytic defect observed in the ∆PQCC mutant could be due to the absence of the N or the C-terminal portions of Ede1 at endocytic sites, rather than the absence of polyQ and coiled-coil domains.

Demonstrating conclusively that Ede1 undergoes a phase transition at endocytic sites and this property is required for endocytic uptake is challenging and will take considerable effort and time. But in essence, these are the points that would actually move the field forward, since phase transitions have been now demonstrated for many proteins, but its functional relevance in many contexts including endocytosis, remains elusive.

We list here a few experiments that could help you prepare a stronger manuscript, but there are likely many more. (1) to hook the wild type Ede1 or the ∆PQCC mutant to an early endocytic protein (i.e. Syp1) in an ede1∆ background and show that the strain expressing the mutant but not the wild type still has an endocytic defect; (2) to finely titrate the concentration of cytosolic Ede1 from 0 to physiological levels and show that the concentration of Ede1 at endocytic sites is sigmoidal rather than linear; (3) to show that another IDR capable of undergoing liquid-phase separation can substitute for the PQ and CC domains of Ede1; or (4) to demonstrate that the PH-PQ-CC construct can recruit the actin machinery and trigger actual endocytic events, but that this capacity is lost under experimental conditions that dissolve the Ede1 condensates (5 min 37oC or 5% hexanediol).

Reviewer #1:

The manuscript addresses what has been a debated issue in the last years regarding the role of intrinsic disordered domains and liquid-liquid phase separation in endocytosis. While it is clear that many endocytic proteins including for example AP180 or Epsins contain this kind of domains, demonstration of a role of liquid-liquid phase separation in the context of membrane budding in vivo has remained elusive. In this work, Kozak and Kaksonen identify a region in Ede1, an early endocytic factor of the yeast S. cerevisiae, which seems to be necessary and sufficient to trigger liquid-liquid phase separation when overexpressed in vivo (based on 4 criteria: the observation of fission and fusion events, fast recovery upon photobleaching, reversible disassembly upon heating and sensitivity to 1,6-hexanediol). Deletion of this region in Ede1 causes endocytic defects, whereas its covalent attachment to a PH domain, which targets it to the plasma membrane, generates cortical patches that recruit other endocytic components and seem to trigger endocytic events.

While the data is interesting, it is still a bit preliminary, and it does not really probe at the moment that the putative liquid-liquid-phase separation induced by this Ede1 region is the relevant feature required for endocytic uptake. The authors would need to take into consideration a few points:

The authors show that the Ede1 foci undergo fusion and fission events and use this observation to support that the Ede1 foci correspond to a separated liquid phase. How often are these events observed? Are those fusion and fission events associated to membranes?

Does Ede1 undergo liquid-liquid phase separation in vitro? The authors mention that this is the case for the mammalian homolog Eps15, but they would need to show that this is the case for Ede1.

Other endocytic proteins have intrinsically disordered domains. How is temperature and 1,6-hexanediol affecting these proteins. Could the authors include controls of other bona-fide non-membrane bound organelles for comparison?

The authors show that deletion of the Ede1 region inducing liquid-liquid phase separation install endocytic defects, but the corresponding construct is not recruited to endocytic sites, so the defects could also be caused by the absence of other Ede1 domains. The authors would need to artificially hook the Ede1 mutant to another early component (i.e. Syp1) and check then if an endocytic defect is installed.

Are the effects of the PH-Ede1 construct, and in particular Sla1 recruitment, especially sensitive to temperature and/or 1,6-hexanediol treatment? It is difficult to evaluate from the kymographs in figure 7 if Sla1 fades away or it is internalized. Can the authors show images of wild type Ede1/Sla1 patches and quantify internalization in both situations? Is Abp1 also recruited to the PH-Ede1 foci? This would be expected if internalization occurs.

Reviewer #2:

Kozak and Kaksonen propose that Ede1 may initiate endocytic events due to its phase separation properties. We found this manuscript very interesting. There's no escaping phase separation right now in cell biology, but this manuscript strikes a nice balance between hype and reality because they are upfront about what is studied here (by overexpression or expression in mutant background) and what it suggests may be happening in normal cells during endocytosis. We think the experiments presented do support the conclusions. Complementary work on bioRxiv shows similar behaviour of the mammalian homologue Eps15, which gives confidence that the authors are on the right track here.

There was one major point that if addressed experimentally (under non-pandemic circumstances) would strengthen the manuscript. The data on the condensates are convincing, but we wondered whether the Ede1 blobs are truly liquid condensates that are devoid of membrane. In our own work in mammalian cells we have seen large blobs of overexpressed protein that turn out to be MVB-like structures by EM. These structures show similar FRAP profiles to the data here, and I don't think any of the experimerts presented here necessarily rule out that these structures may have a membrane component.

Our remaining points are straightforward to address with existing data and would help to improve the paper.

1. Figure 1D number of cells with condensates is annotated. Are there differences in the number or brightness of condensates between EDE1-EGFP/EDE1-EGFP and EDE1-EGFP/EDE1?

2. Figure 2C would be improved by including the quantification that is listed in the text i.e. graph with average and fit.

3. Figure 3 who recruits who in this figure? It is convincing that the blob is due to Ede1 but the site of the blob could be dictated by any one of the other proteins. Do movies of blob formation show that each protein appears after Ede1 or do they simultaneously accumulate?

4. Figure 4C shows FL gives 100% of cells with condensates but the percentage is lower in Figure 1D – is this just experimental variability?

5. Figure 5CD is there a correlation between patch density and lifetime? If they are plotted against each other do they scale linearly? Also, the movies in the manuscript work well and for this figure, a side-by-side movie of wt, ∆PQCC and ede1∆ would be a useful addition.

6. Figure 6 shows a representative cell. Quantification would strengthen this figure.

Reviewer #3:

In this paper, Kozak and Kaksonen report that the budding yeast's early endocytic protein Ede1, a homologue of mammalian Esp15 can form aggregates when the protein is overexpressed or expressed in absence of 3 other early endocytic proteins (the AP180 family proteins Yap1801/2 and the AP2 complex α-subunit Apl3). The paper aims at showing that these aggregates are phase separated structures and speculate that phase separation could be the mode of assembly of Ede1 during clathrin-mediated endocytosis in yeast. The authors also identified the minimum portion of Ede1 that leads to these aggregates.

1. The data are relatively solid to demonstrate that the aggregates seen in the mutants can be phase separated structures. However, I am not convinced the data strongly support that Ede1 experiences phase separation at sites of clathrin-mediated endocytosis. To my opinion, several pieces of data strongly suggest that Ede1 assembles into two different types of structures in the mutant strains – one structure at the endocytic sites, which is due to specific high affinity interactions, and one structure at the aggregates that is partly due to new low affinity interactions that are not present in normal conditions at the endocytic sites (possibly in addition to the other high affinity interactions mentioned above). Indeed, several of the key experiments that aim to show phase separation yield different results for the aggregates and the endocytic sites:

(1a) Aggregates and endocytic sites are disassembled at different temperature. This is expected from the laws of thermodynamics if the interactions in aggregates and endocytic sites have different affinities. Indeed, the Kd of any reversible biochemical interaction depends exponentially on the temperature and low affinity interactions (from the aggregates) will become unfavorable at lower temperature than higher affinity interactions (from the endocytic sites).

(1b) 1,6-hexanediol dissociates aggregates at lower concentrations than it dissociates endocytic patches, which also suggest the interactions in the aggregates have lower affinity. As a side note, I want to point out that even though researcher in the phase separation field routinely use hexanediol to demonstrate phase separation, it remains somewhat controversial [McSwiggen et al. 2019].

(1c) FRAP on endocytic sites does not demonstrate that they are phase separated structures: many other endocytic proteins do exchange rapidly and are not believed to be phase separated (e.g. the actin cytoskeleton proteins [Kaksonen et al. 2003, Kaksonen et al. 2005, Lacy et al. 2019]).

(1d) The mobile and immobile fractions are different in aggregates and endocytic sites.

2. Since Ede1 concentration seems to be critical for the formation of the aggregates, it is important to systematically report the cytosolic and cell concentrations (or even the relative intensities) in different experimental conditions:

(2a) l. 100, it is suggested that the levels of cytoplasmic Ede1 might be different in wild-type and the triple mutant. Concentrations (even relative) should be reported.

(2b) In Figure 4 it is important to make sure the aggregation with different constructs is not due to differences in the cytoplasmic or cell concentrations of the constructs.

(2c) Figure 7C and l. 252+: how the behavior of the puncta formed with the Ede1(366-900)-2xPH construct depend on concentration? Also, do all the puncta recruit Sla1? Is Sla1 also recruited at sites where the Ede1 construct is not present?

3. l. 157: I am not sure I understand the logic (I may have missed something). The fact that it is a condensate does not imply it triggers actin polymerization. This is a correlation, not a causation. In addition, one could imagine there is a defined endocytic patch within a diffraction limited distance from the condensate. Quantifying the amount of abp1 (relatively to other patches) could be a way to tell whether these are bona fide endocytic structures coming out of the aggregate.

4. In conclusion, in its current form, this study does convince me that Ede1 phase separates during clathrin-mediated endocytosis. In addition, it is unclear to me how the putative phase separation of Ede1 would affect endocytosis, compared to regular protein assembly. I believe several extra experiments would be necessary to make a strong point.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Thank you for submitting your article "Phase separation of Ede1 promotes the initiation of endocytosis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Stephen J Royle (Reviewer #2).

The manuscript addresses what has been a debated issue in the last years, regarding the role of intrinsic disordered regions (IDRs) and liquid phase separation in endocytosis. While it is clear that many endocytic proteins, including AP180 or Epsins, contain this kind of sequences, conclusive demonstration of a role of liquid phase separation in the context of membrane budding in vivo has remained elusive. In this work, Kozak and Kaksonen identify a region in the yeast Eps15 homolog Ede1 (comprising a coiled-coil domain (CC) and a PQ rich region (PQ)), which is necessary and sufficient to trigger liquid phase separation when overexpressed in vivo (based on 4 criteria: the observation of fission and fusion events, fast recovery upon photo-bleaching, reversible disassembly upon heating and sensitivity to 1,6-hexanediol). Deletion of this region in Ede1 causes endocytic defects, whereas its covalent attachment to a PH domain, which targets it to the plasma membrane, generates cortical patches that recruit other endocytic components. Strikingly also, IDRs from other unrelated proteins known to trigger liquid phase separation can partially complement the Ede1 mutant lacking the CC and PQ regions.

While the data is of high quality and consistent with the role of liquid phase separation in endocytosis, other models might as well explain the data. The ectopic IDRs might work as linkers or spacers of the N and C-terminal domains, or they might promote oligomerization. In the context of this debated hypothesis, the other possibilities need to be tested. Also, the requirement for the CC and PQ regions in endocytosis is not conclusive because the mutant lacking these domains is not recruited to endocytic sites, so the endocytic defect might as well be caused by the absence of the N or C-terminal domains.

After some discussion on the new version of the manuscript, there is a general agreement on the interest of the article and the quality of the data. However, two out of three reviewers still consider that the functional relevance of liquid phase separation in endocytosis is not conclusively proven and other hypothesis might as well explain the data. Even though the observation that other unrelated Intrinsically Disorder Regions (IDRs) can partially substitute for the Ede1 regions promoting liquid phase separation is quite compelling, the inserted IDRs might as well work by providing a flexible loop of a given size between the N and C terminal domains or by promoting oligomerization. Since the role of liquid phase separation in endocytosis is under strong debate, we feel that these two other possibilities need to be properly tested and discussed before publication. Even if the new data would disprove the liquid phase separation model, the article would still be of interest for eLife. If the new oligomerization or linker regions cannot functionally substitute for the Ede1 coiled-coil (CC) and PQ rich (PQ) regions, the data would provide stronger support for the role of liquid phase separation in endocytosis but still, the title, the abstract and the discussion would need to be rephrased to avoid overstatements that would slow down advance in the field. In addition to this main concern, there is still the issue on the recruitment of the Ede1-(PQ,CC)∆ mutant to endocytic sites. At this point, it is not possible to conclude that the CC-PQ region is required for endocytic uptake because the mutant is not recruited to endocytic sites. One of the reviewers also suggested to investigate the possible differences in the posttranslational modifications that could lead to phase-separation. Even though this approach could be enlightening, it will take more than two months and it is probably beyond the scope of the manuscript. In essence then, the manuscript would be adequate for publication in eLife, provided than you address the following key points:

1. Substitute the CC and PQ regions for non-IDRs that can act as linkers or oligomerization domains and investigate if the chimeras can complement the ede1∆ phenotype.

2. Solve the issue of the recruitment of the Ede1-(PQ,CC)∆ to endocytic sites.

3. Rewrite the title, the abstract and the discussion to make clear that the results support the requirement for liquid-phase separation in endocytosis but they do not conclusively prove it.

In addition to these main concerns, please carefully read the rest of the points raised by the reviewers and discuss or address them:

1. The new data showing Ede1 concentration buffering is interesting. However, we find the authors' interpretation unclear at places and, to our understanding, only one possible interpretation of the data. The authors say the cells control their cytoplasmic concentration of Ede1 but, to them, is it an active or a passive process? For bona fide purely phase separating proteins, this can be a passive process (once phase separation happens, the soluble concentration remains virtually constant and the size of the condensates changes with changing concentration). The data presented in the paper is indeed compatible with this hypothesis. However, the data is also compatible with equally likely alternative hypotheses. One is that there are two populations of Ede1 with different post-translational modifications because of the overexpression. As the authors point out, Ede1 is the most heavily phosphorylated protein in CME (Lu et al. 2016), and it is also ubiquitinated. Therefore, it is possible that overexpressing Ede1 overloads its kinases, phosphatases and/or ubiquitination machineries, and the excess mis-phosphorylated/mi-ubiquitinated proteins would phase separate, without being involved in endocytosis. It is also possible that Ede1 does not normally phase separate at endocytosis sites because it is bound to endocytic partners or cargo. Overexpressing Ede1 would create a stoichiometric imbalance at endocytic sites and the excess protein would end up in the cytoplasm and could phase separate there only.

2. We find it surprising that the FUS domain does not phase separate Ede1 since FUS is a well known phase separating domain, and other phase separating domains seem to work. Do the authors have an explanation for that?

3. L 208: "catalyzes cycles of assembly and disassembly of actin": it is not clearly demonstrated, so this could reformulated.

4. L 392: "likely": change to "possibly" because this is not demonstrated in the paper (just correlation with abp1).

5. In response to reviewer #2, the authors point out that hexanediol causes the PH domain to form clusters, but say they are not showing it in the paper because it is only peripherally related to the paper. I agree but this could be useful for the field to report this anyway because there are a lot of controversies about the use of hexanediol in the phase separation field.

6. Talking about fusion and fission of condensates is an overstatement. It might just be the condensates getting closer or separating. Talking about apparent fission or fusion events throughout the text would be more accurate. How often are the putative fission events observed?

7. Is the PQ region alone generating condensates? The authors test the 1-591 Ede1 region containing the EH domains and the PQ region, but not the PQ region alone. The EH domain might somehow inhibit the formation of condensates by the IDR.

8. Are the condensates co-localizing FM4-64 stainable membranes? If so, the membrane association might provide for the liquid-phase-like behavior.

9. Does Abp1 assemble on all condensates on the time-lapse movies? Does the transient Abp1 assembly on the condensates explain the low percentage of co-localization? Are condensates hot spots for Abp1 assembly?

10. Are the GFP-Ede1(366-900) condensates behaving as the GFP-Ede1 condensates in terms of temperature and hexanodiol sensitivity? It is surprising that the GFP- Ede1(366-900)-2xPH condensates do not. If the GFP-Ede1(366-900) condensates behave as separate liquid-phase condensates, similar to those generated by Ede1, it might mean that their membrane association modifies the Ede1-liquid-phase properties. In this context, are the PM associated Ede1 condensates (dome-shaped) behaving similar to those assembled within the cytosol?

11. It is still not so clear if the GFP- Ede1(366-900)-2xPH can really initiate bona fide endocytic events. The internalization of the Sla1 patches is not clear in the images provided. If they are bona fide endocytic events, the fact that GFP- Ede1(366-900)-2xPH condensates do not behave as a liquid-phase would suggest that the formation of an Ede1-dependent liquid phase is not really essential for endocytic uptake and that the Ede1 central region rather operates as a linker or by recruiting other endocytic proteins.

12. The Rapamycin inducible system might not be the most adequate to bring the Ede1 trucations to endocytic sites because the tor1 mutations used to make the yeast strain resistant to Rapamycin might interfere with endocytic uptake. Are Syp1 and Sla1 cortical patch dynamics similar to the wild type´s in a tor1-1 frp1 EDE1-mCherry-FKPB SYP1-FRB background? It might be easier to covalently link the Ede1 truncations to Syp1 or to a later endocytic coat component that is less affected by depletion of Ede1.

Reviewer #1:

Kozak and Kaksonen demonstrate that the mammalian Eps15 yeast homolog Ede1 can phase-separate upon overexpression or deletion of endocytic adaptors in vivo. The authors define the protein regions necessary and sufficient to generate the condensates and demonstrate that those are probably necessary and sufficient to sustain the endocytic function of Ede1. Conclusive demonstration on whether liquid phase-separation actually occurs at endocytic sites in wild type cells remains elusive. However, the authors show that unrelated protein sequences known to undergo this transition can functionally (albeit partially) substitute for the Ede1 region generating the condensates.

Even though the data points to the functional role of a separated Ede1 liquid phase in endocytic uptake, some important issues are still unsolved:

1. The authors conclude that the coiled-coil and IDR of Ede1 is required for endocytic uptake because using a Rapamycin inducible system to hook the Ede1 mutant lacking these domains to Syp1, does not complement the ede1∆ defects. Again, this particular experiment is not very informative because the Ede1 mutant is not really recruited to endocytic sites in the presence of Rapamycin and therefore, the endocytic defect might be because by the absence of the N or C-terminal domains. The Rapamycin inducible system might not be the most adequate to bring the Ede1 trucations to endocytic sites because the tor1 mutations used to make the yeast strain resistant to Rapamycin might interfere with endocytic uptake. Are Syp1 and Sla1 cortical patch dynamics similar to the wild type´s in a tor1-1 frp1 EDE1-mCherry-FKPB SYP1-FRB background? It might be easier to covalently link the Ede1 truncations to Syp1.

Other important but easily addressable points:

1. Talking about fusion and fission of condensates is an overstatement. It might just be the condensates getting closer or separating. Talking about apparent fission or fusion events throughout the text would be more accurate. How often are the putative fission events observed?

2. Is the PQ region alone generating condensates? The authors test the 1-591 Ede1 region containing the EH domains and the PQ region, but not the PQ region alone. The EH domain might somehow inhibit the formation of condensates by the IDR.

3. Are the condensates co-localizing FM4-64 stainable membranes? If so, the membrane association might provide for the liquid-phase-like behavior.

4. Does Abp1 assemble on all condensates on the time-lapse movies? Does the transient Abp1 assembly on the condensates explain the low percentage of co-localization? Are condensates hot spots for Abp1 assembly?

5. Are the GFP-Ede1366-900 condensates behaving as the GFP-Ede1 condensates in terms of temperature and hexanodiol sensitivity? It is surprising that the GFP- Ede1366-900-2xPH condensates do not. If the GFP-Ede1366-900 condensates behave as separate liquid-phase condensates, similar to those generated by Ede1, it might mean that their membrane association modifies the Ede1-liquid-phase properties. In this context, are the PM associated Ede1 condensates (dome-shaped) behaving similar to those assembled within the cytosol?

6. It is still not so clear if the GFP- Ede1366-900-2xPH can really initiate bona fide endocytic events. The internalization of the Sla1 patches is not clear in the images provided. If they are bona fide endocytic events, the fact that GFP- Ede1366-900-2xPH condensates do not behave as a liquid-phase would suggest that the formation of an Ede1-dependent liquid phase is not really essential for endocytic uptake and that the Ede1 central region rather operates as a linker or by recruiting other endocytic proteins.

Reviewer #2:

The authors have addressed all the points that I raised in my original review. I feel that the new work added has strengthened the manuscript.

During the last review, I was quite positive about this paper; the other reviewers and editors less so. I share the general scepticism about "phase separation explains all cell biology" hype, however I think this is a careful study and the authors are quite conservative in their interpretation.

Since this manuscript was posted it has been cited 10 times (according to Google Scholar today) suggesting the community are finding it useful. I also note the companion preprint by Day et al. (showing similar results for the human homolog) was published in Nature Cell Biology back in April. I don't think delaying publication of this manuscript further is helping anyone.

Reviewer #3:

We thank the authors for their efforts in addressing our initial concerns and providing new data. We think the paper has been improved since the first submission. However, we think there are still important remaining issues.

1. The authors provide a more thorough characterization of the phase separated Ede1 structures observed in mutant cells or after over-expression. However, we are still not completely convinced phase separation does happen during endocytosis and is functionally important for this process. We agree with the authors that all the data are "compatible" with this idea, but they are also compatible with the idea that phase separation is a protection mechanism for excess protein or non-functional proteins. The authors acknowledge that in the response to reviewers ("we cannot exclude the possibility that the condensation of some constructs in the wild-type background is due to the truncations or the increase in protein levels") and use "likely" very often in their response when talking about phase separation during endocytosis. Most arguments we used in our first review are still valid even with the new data. We acknowledge that completely disproving the alternative hypotheses will be tedious but at least the text should present more clearly that several interpretations are compatible with the data, and the title and abstract should be toned down.

2. The new data showing Ede1 concentration buffering is interesting. However, we find the authors' interpretation unclear at places and, to our understanding, only one possible interpretation of the data. The authors say the cells control their cytoplasmic concentration of Ede1 but, to them, is it an active or a passive process? For bona fide purely phase separating proteins, this can be a passive process (once phase separation happens the soluble concentration remains virtually constant and the size of the condensates changes with changing concentration). The data presented in the paper is indeed compatible with this hypothesis. However, the data is also compatible with equally likely alternative hypotheses. One is that there are two populations of Ede1 with different post-translational modifications because of the overexpression. As the authors point out, Ede1 is the most heavily phosphorylated protein in CME (Lu et al. 2016), and it is also ubiquitinated. Therefore, it is possible that overexpressing Ede1 overloads its kinases, phosphatases and/or ubiquitination machineries, and the excess mis-phosphorylated/mi-ubiquitinated proteins would phase separate, without being involved in endocytosis. It is also possible that Ede1 does not normally phase separate at endocytosis sites because it is bound to endocytic partners or cargo. Overexpressing Ede1 would create a stoichiometric imbalance at endocytic sites and the excess protein would end up in the cytoplasm and could phase separate there only.

3. The new data where the PQCC domains are replaced with known phase separating domains is nice and interesting. We are wondering if phase separation is required or if strong dimerization or oligomerization would be sufficient for the rescue? This hypothesis is compatible with the fact that the effects of the ΔCC and ΔPQ deletions seem additive and that the ΔCC (possibly a dimerization domain) mutant has very weak localization to endocytic patches. To test this hypothesis, the authors could try to rescue the ΔCC, ΔPQ and ΔPQCC mutants with dimerization or oligomerization domains, instead of phase separating domains.

https://doi.org/10.7554/eLife.72865.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

In summary, there is a consensus on the importance of the scientific question addressed by your work, and on the nature of the Ede1 condensates when it is overexpressed or in the endocytic adaptor mutant background. However, 2 of 3 reviewers considered that the manuscript does not demonstrates that Ede1 really undergoes liquid-liquid phase separation at endocytic sites under physiological conditions and whether this property is required for its endocytic function. The concerns follow.

We now describe several of the suggested experiments, as well as some which were not suggested but, in our view, significantly strengthen the manuscript. We also rewrote the Discussion section to more effectively ground our findings in the broader context of endocytic assembly and the evolving understanding of phase separation processes.

1. Several of the key experiments aimed at showing phase separation yield different results for the condensates and the endocytic sites. Basically, they disassemble at different temperatures and at different hexanediol concentrations. Together with the fact that FRAP per se does not demonstrate that Ede1 forms a separate liquid phase at endocytic sites, the data rather suggest that the Ede1 condensates and the Ede1 endocytic patches have different properties.

These are valid concerns, but we do not see these results as contradictory. We do not wish to imply that phase separation is the sole driver of endocytosis, but rather, that Ede1 promotes endocytosis through a mechanism consistent with the phase separation framework. The machinery found at bona fide endocytic sites is of course complex and contains many types of interactions, not all of which can be found in abnormal Ede1 condensates. Importantly, we now show that, under normal wild type conditions, cells maintain the cytosolic Ede1 level at the critical concentration for phase separation (new Figure 3). Therefore, even in normal cells Ede1 is poised to undergo phase separation.

We rewrote several portions of the text, including most of the Discussion, to convey our reasoning with more clarity and to highlight how assembly via phase separation is distinct from other mechanisms of assembly.

2. The experiment showing that domains required to form the condensates are required for endocytosis is not really conclusive because, among other considerations, there seems to be an inverse correlation between the capacity of the different mutants to be recruited at endocytic sites and their capacity to sustain endocytosis. Under these circumstances, the endocytic defect observed in the ∆PQCC mutant could be due to the absence of the N or the C-terminal portions of Ede1 at endocytic sites, rather than the absence of polyQ and coiled-coil domains.

We agree that the terminal regions of Ede1 are also important for endocytosis. After all, the central region alone is also not efficiently recruited to endocytic sites, forming instead intracellular condensates.

However, we do not see this as a contradiction. To the best of our knowledge, the N- and C- terminal regions harbour interaction motifs and not catalytic domains. In our model, the primary function of Ede1 is to concentrate itself (through phase separation of the self-interacting central region) and other proteins (through the N- and C-terminal interaction domains). If Ede1 cannot form patches at the plasma membrane as a consequence of ∆PQCC mutation, it cannot perform this function.

Nevertheless, we performed the experiment suggested below, and found that artificial recruitment of the ∆PQCC mutant to other endocytic proteins cannot rescue its function.

Demonstrating conclusively that Ede1 undergoes a phase transition at endocytic sites and this property is required for endocytic uptake is challenging and will take considerable effort and time. But in essence, these are the points that would actually move the field forward, since phase transitions have been now demonstrated for many proteins, but its functional relevance in many contexts including endocytosis, remains elusive.

We list here a few experiments that could help you prepare a stronger manuscript, but there are likely many more.

(1) to hook the wild type Ede1 or the ∆PQCC mutant to an early endocytic protein (i.e. Syp1) in an ede1∆ background and show that the strain expressing the mutant but not the wild type still has an endocytic defect;

We have now performed this experiment using the anchor-away inducible dimerization technique with Ede1 and Syp1. Neither the localization of the Ede1 mutant nor Sla1 patch density change significantly after rapamycin addition (Figure 6—figure supplement 1). The recruitment of Ede1 EH domains and C-terminus to membrane-binding endocytic proteins is thus insufficient to promote endocytosis on its own.

(2) to finely titrate the concentration of cytosolic Ede1 from 0 to physiological levels and show that the concentration of Ede1 at endocytic sites is sigmoidal rather than linear;

We believe that the experiment suggested here, while potentially very valuable, would pose significant technical challenges, from titrating the in vivo concentration finely enough to imaging the earliest stages of site formation with sufficient sensitivity to support this kind of analysis. Furthermore, we don’t think that the concentration of Ede1 at the endocytic sites is solely determined by a phase separation like process, but also via more “classical” interactions with the lattice of the endocytic coat.

This suggestion however, led us to test a related prediction of phase separation, namely the existence of a critical protein concentration in the cytosol (new Figure 3). We measured the cytosolic and total cellular fluorescence intensity of tagged Ede1 and found that the cytosolic concentration does not increase above wild-type levels even in cells in which Ede1 is over-expressed. This suggests that the normal cytosolic concentration of Ede1 is buffered by phase separation, and that the excess Ede1 is condensed at endocytic sites, and at higher expression levels into the Ede1 condensates. The fact that cells maintain cytosolic Ede1 at the critical concentration means that the phase separation activity of Ede1 is highly likely to contribute to its normal assembly at the endocytic sites.

(3) to show that another IDR capable of undergoing liquid-phase separation can substitute for the PQ and CC domains of Ede1; or

We substituted four different low-complexity domains of different lengths for the central region of Ede1 (new Figure 8). The domains were chosen based on criteria such as prion-like sequence and known tendency to phase separate. The replacements partially rescue the localization and function of Ede1. Although we couldn’t fully re-engineer the complete functionality, we think this provides support for phase separation activity contributing to Ede1’s normal function.

(4) to demonstrate that the PH-PQ-CC construct can recruit the actin machinery and trigger actual endocytic events, but that this capacity is lost under experimental conditions that dissolve the Ede1 condensates (5 min 37oC or 5% hexanediol).

The experiment presented in Figure 9 (former Figure 7) was meant to merely illustrate the ability of Ede1’s central region to concentrate membrane-bound proteins. Finding that it interacts with the endocytic machinery was unexpected, and we think that the full characterization of this interaction is beyond the scope of this article.

Reviewer #1:

The manuscript addresses what has been a debated issue in the last years regarding the role of intrinsic disordered domains and liquid-liquid phase separation in endocytosis. While it is clear that many endocytic proteins including for example AP180 or Epsins contain this kind of domains, demonstration of a role of liquid-liquid phase separation in the context of membrane budding in vivo has remained elusive. In this work, Kozak and Kaksonen identify a region in Ede1, an early endocytic factor of the yeast S. cerevisiae, which seems to be necessary and sufficient to trigger liquid-liquid phase separation when overexpressed in vivo (based on 4 criteria: the observation of fission and fusion events, fast recovery upon photobleaching, reversible disassembly upon heating and sensitivity to 1,6-hexanediol). Deletion of this region in Ede1 causes endocytic defects, whereas its covalent attachment to a PH domain, which targets it to the plasma membrane, generates cortical patches that recruit other endocytic components and seem to trigger endocytic events.

While the data is interesting, it is still a bit preliminary, and it does not really probe at the moment that the putative liquid-liquid-phase separation induced by this Ede1 region is the relevant feature required for endocytic uptake. The authors would need to take into consideration a few points:

The authors show that the Ede1 foci undergo fusion and fission events and use this observation to support that the Ede1 foci correspond to a separated liquid phase. How often are these events observed? Are those fusion and fission events associated to membranes?

We did not quantify the exact frequency of fission or fusion events as these are regular, but relatively rare. A rough estimate would be that in a field of a few dozen cells, we will usually observe one such event per minute.

As the condensates are frequently associated with membranes, the fission and fusion events are as well, but we have also seen free-floating condensates split and/or fuse back.

Does Ede1 undergo liquid-liquid phase separation in vitro? The authors mention that this is the case for the mammalian homolog Eps15, but they would need to show that this is the case for Ede1.

We agree that in vitro experiments would provide valuable insights. However, we believe that we have provided a substantial amount of in vivo evidence. Establishing a protein purification pipeline would significantly delay publication of the in vivo data.

Other endocytic proteins have intrinsically disordered domains. How is temperature and 1,6-hexanediol affecting these proteins. Could the authors include controls of other bona-fide non-membrane bound organelles for comparison?

The effects of 1,6-hexanediol indeed apply to a wide range of membraneless organelles (Kroschwald et al., 2015; Molliex et al., 2015; Nott et al., 2015). We chose to focus on other suggested experiments rather than repeat the hexanediol treatments found in literature. We discuss the non-specific nature of hexanediol treatment in the manuscript and note that it is only one of several lines of evidence.

Other authors have shown that late coat proteins like Sla1 are also susceptible to dissolution by temperature, but at higher values than Ede1 (Bergeron-Sandoval et al., 2017). Our experience with Sla1 confirms that it is more stable than Ede1. A full screen for the sensitivity of endocytic proteins to temperature treatments would be an interesting idea but outside of the scope of this manuscript.

The authors show that deletion of the Ede1 region inducing liquid-liquid phase separation install endocytic defects, but the corresponding construct is not recruited to endocytic sites, so the defects could also be caused by the absence of other Ede1 domains. The authors would need to artificially hook the Ede1 mutant to another early component (i.e. Syp1) and check then if an endocytic defect is installed.

We have now performed the suggested experiment by attaching Ede1∆PQCC-mCherry-FRB to Syp1-FKBP via rapamycin-induced dimerization. We did not observe a rescue of the patchy Ede1 localization pattern. We also quantified the number of Sla1-EGFP sites with and without rapamycin and detected no statistically significant increase in Sla1 density (Figure 6—figure supplement 1). We conclude that the mere recruitment of Ede1 N- and C-terminal regions to other membrane interacting endocytic proteins is insufficient to promote the initiation of endocytosis.

Are the effects of the PH-Ede1 construct, and in particular Sla1 recruitment, especially sensitive to temperature and/or 1,6-hexanediol treatment?

We abandoned the idea of using 1,6-hexanediol on the Ede1PQCC-2xPH construct during preliminary experiments. We found that hexanediol treatment causes the PH domain itself to form clusters, or possibly membrane invaginations. This finding is another demonstration of the wide-ranging effects of hexanediol on living cells. While we think it is only peripherally related to the topic of the article and thus did not include it, we can add the data if requested.

To address this question, we have however added a FRAP experiment which shows that the structures formed by the Ede1PQCC-2xPH construct have a solid-like, rather than liquid-like, state (Figure 9—figure supplement 1). We discuss this observation on line 483 of the manuscript: “However, the GFP-Ede1PQCC-2×PH puncta persist over long imaging periods and do not disassemble after Sla1 internalization. Structures formed by GFP-Ede1PQCC-2×PH also do not recover fluorescence. This suggests that while the central region of Ede1 can drive phase separation, the terminal regions are needed to maintain the liquid state.”

It is difficult to evaluate from the kymographs in figure 7 if Sla1 fades away or it is internalized. Can the authors show images of wild type Ede1/Sla1 patches and quantify internalization in both situations? Is Abp1 also recruited to the PH-Ede1 foci? This would be expected if internalization occurs.

The kymographs in Figure 9 (formerly Figure 7) were generated from TIRF movies (Figure 9—video 1) and therefore cannot show inward movement, which happens along the optical axis. However, the equatorial plane movie shows inward movement of Sla1 patches (Figure 9—video 2).

Reviewer #2:

Kozak and Kaksonen propose that Ede1 may initiate endocytic events due to its phase separation properties. We found this manuscript very interesting. There's no escaping phase separation right now in cell biology, but this manuscript strikes a nice balance between hype and reality because they are upfront about what is studied here (by overexpression or expression in mutant background) and what it suggests may be happening in normal cells during endocytosis. We think the experiments presented do support the conclusions. Complementary work on bioRxiv shows similar behaviour of the mammalian homologue Eps15, which gives confidence that the authors are on the right track here.

There was one point that if addressed experimentally (under non-pandemic circumstances) would strengthen the manuscript. The data on the condensates are convincing, but we wondered whether the Ede1 blobs are truly liquid condensates that are devoid of membrane. In our own work in mammalian cells we have seen large blobs of overexpressed protein that turn out to be MVB-like structures by EM. These structures show similar FRAP profiles to the data here, and I don't think any of the experimerts presented here necessarily rule out that these structures may have a membrane component.

This is a reasonable hypothesis that we had already planned on addressing with electron microscopy. However, during the course of preparing and revising the manuscript, other authors published independent, complementary work investigating the removal of endocytic protein condensates via Ede1-mediated autophagy (Wilfling et al., 2020). They performed correlated cryo-ET experiments on the Ede1 droplets in overexpression yeast strains and confirmed that there was no vesicle component in the condensates. Rather, the endocytic condensate appears as a large ribosome exclusion zone comparable to that seen around normal endocytic sites (Kukulski et al., 2012). Wilfling et al. also noted that the endocytic condensates were frequently contacted, but not completely enclosed by, ribosome-containing tubular membranes assumed to be the ER.

Our remaining points are straightforward to address with existing data and would help to improve the paper.

1. Figure 1D number of cells with condensates is annotated. Are there differences in the number or brightness of condensates between EDE1-EGFP/EDE1-EGFP and EDE1-EGFP/EDE1?

As expected, heterozygous cells with only one tagged Ede1 allele are on the whole dimmer than the homozygotes, as both the sites and the condensates in heterozygous cells contain some amount of tagged and untagged Ede1. We did not notice any difference in number.

2. Figure 2C would be improved by including the quantification that is listed in the text i.e. graph with average and fit.

We thank the reviewer for the suggestion. We provided this quantification in panel 2D.

3. Figure 3 who recruits who in this figure? It is convincing that the blob is due to Ede1 but the site of the blob could be dictated by any one of the other proteins. Do movies of blob formation show that each protein appears after Ede1 or do they simultaneously accumulate?

Because the cells are genomic knockouts (triple deletion) or constitutive overexpression, and the condensates persist throughout the cell cycle (Figure 1—video 1), we do not actually have movies of de novo condensate formation.

An exception is the new experiment with galactose-induced Ede1 overexpression in new Figure 3 (see Figure 3—video 1 in particular), but in these cells, only Ede1 was fluorescently tagged. The condensates in this experiment do initially appear as normal endocytic sites but end up growing instead of progressing into movement and disassembly.

4. Figure 4C shows FL gives 100% of cells with condensates but the percentage is lower in Figure 1D – is this just experimental variability?

We added more quantifications to provide a better idea of the variability present in these experiments. The strains in question are not the same (Figure 1D is diploid, 4C is haploid) but from the data we have, it does not appear they are significantly different in terms of the fraction of cells with Ede1 condensates.

5. Figure 5CD is there a correlation between patch density and lifetime? If they are plotted against each other do they scale linearly? Also, the movies in the manuscript work well and for this figure, a side-by-side movie of wt, ∆PQCC and ede1∆ would be a useful addition.

The plot in Author response image 1 shows mean lifetime and density values and their 95% confidence interval (based on the inter-experimental variability). There is a fairly robust correlation (adjusted R2 = 0.93), shown is a trend line with 95% confidence interval (shaded area).

Author response image 1

We carefully considered adding more movies to the manuscript. In this case, the phenotype is subtle and requires careful quantification. We believe that the movies on their own would not be illustrative enough to the readers to warrant their inclusion.

6. Figure 6 shows a representative cell. Quantification would strengthen this figure.

We completely agree with the reviewer. However, the phenotype presented in Figure 7 (formerly Figure 6) is one of a reduced tendency of early adaptors to cluster – ‘spotty’ vs ‘fuzzy’ localization at the sites. This is unlike the phenotype presented in Figure 6 (formerly Figure 5), where Sla1 sites are reduced in number, but retain good contrast for thresholding and counting. In addition, the phenotypes are similar, but not the same for all proteins. For example, Syp1 loses the punctate localization on the plasma membrane, but remains bright at the bud necks, where it localizes independently of Ede1.

We were thus unable to find a measure of signal dispersion that would be meaningful and generalizable to all strains presented in this figure, and chose to present the result in a qualitative manner.

Reviewer #3:

In this paper, Kozak and Kaksonen report that the budding yeast's early endocytic protein Ede1, a homologue of mammalian Esp15 can form aggregates when the protein is overexpressed or expressed in absence of 3 other early endocytic proteins (the AP180 family proteins Yap1801/2 and the AP2 complex α-subunit Apl3). The paper aims at showing that these aggregates are phase separated structures and speculate that phase separation could be the mode of assembly of Ede1 during clathrin-mediated endocytosis in yeast. The authors also identified the minimum portion of Ede1 that leads to these aggregates.

1. The data are relatively solid to demonstrate that the aggregates seen in the mutants can be phase separated structures. However, I am not convinced the data strongly support that Ede1 experiences phase separation at sites of clathrin-mediated endocytosis. To my opinion, several pieces of data strongly suggest that Ede1 assembles into two different types of structures in the mutant strains – one structure at the endocytic sites, which is due to specific high affinity interactions, and one structure at the aggregates that is partly due to new low affinity interactions that are not present in normal conditions at the endocytic sites (possibly in addition to the other high affinity interactions mentioned above). Indeed, several of the key experiments that aim to show phase separation yield different results for the aggregates and the endocytic sites:

(1a) Aggregates and endocytic sites are disassembled at different temperature. This is expected from the laws of thermodynamics if the interactions in aggregates and endocytic sites have different affinities. Indeed, the Kd of any reversible biochemical interaction depends exponentially on the temperature and low affinity interactions (from the aggregates) will become unfavorable at lower temperature than higher affinity interactions (from the endocytic sites).

Ede1 has many different known interactions: both self-interaction via its core region and several interactions with various other endocytic proteins via its N- and C-terminal domains. It is highly likely that these interaction networks are not identical between endocytic sites and the condensates leading to different affinities. We don’t think that the different affinities are contradicting our conclusions.

(1b) 1,6-hexanediol dissociates aggregates at lower concentrations than it dissociates endocytic patches, which also suggest the interactions in the aggregates have lower affinity. As a side note, I want to point out that even though researcher in the phase separation field routinely use hexanediol to demonstrate phase separation, it remains somewhat controversial [McSwiggen et al. 2019].

We completely agree with the reviewer as to the controversial nature of 1,6-hexanediol. It is known to have unexpected systemic side-effects (Kroschwald et al., 2017) and long treatments can even induce stress granule formation (Wheeler et al., 2016). We are aware of these limitations, which is why (a) the duration of our experiments with hexanediol was kept to a minimum, and (b) it is only one of several applied criteria. We now state this explicitly in the text.

(1c) FRAP on endocytic sites does not demonstrate that they are phase separated structures: many other endocytic proteins do exchange rapidly and are not believed to be phase separated (e.g. the actin cytoskeleton proteins [Kaksonen et al. 2003, Kaksonen et al. 2005, Lacy et al. 2019]).

We agree again with the reviewer: our FRAP data at endocytic sites is consistent with phase separation but does not conclusively prove it. We now discuss the technical challenges of testing phase separation at endocytic sites in the Discussion section. However, we believe that in this revised manuscript we have provided data to argue that phase separation of Ede1 is highly likely to take place at the endocytic sites. Particularly, we now show that the cytosolic concentration of Ede1 in wild type cells is maintained at the critical concentration for phase separation. Therefore, it is expected that any additional Ede1, which in normal cells is accumulated at endocytic sites, is poised to phase separate.

(1d) The mobile and immobile fractions are different in aggregates and endocytic sites.

Phase separated condensates are known to exist in different states from highly fluid to gel-like to solid. It is conceivable that the fluidity of Ede1 condensates is modulated by different cellular activities such as different interactions, or post-translational modifications such as phosphorylation or ubiquitination.

2. Since Ede1 concentration seems to be critical for the formation of the aggregates, it is important to systematically report the cytosolic and cell concentrations (or even the relative intensities) in different experimental conditions:

(2a) l. 100, it is suggested that the levels of cytoplasmic Ede1 might be different in wild-type and the triple mutant. Concentrations (even relative) should be reported.

We thank the reviewer for this excellent suggestion. We now quantify the cytosolic and total cell concentrations (new Figure 3). We found that there is a limit to the cytosolic concentration of Ede1, which is in line with the predictions made by a model of phase separation driven primarily by homotypic interactions. Importantly, this limit is reached by wild-type cells, suggesting that Ede1 phase separates in normal conditions.

We also show that the total Ede1 levels are mildly increased in the triple deletion mutant (Figure 3, Figure 5—figure supplement 1).

(2b) In Figure 4 it is important to make sure the aggregation with different constructs is not due to differences in the cytoplasmic or cell concentrations of the constructs.

We have now addressed this question by western blotting (Figure 5—figure supplement 1). All of the concentrations of truncated constructs are elevated compared to the full-length in wild-type background cells.

At this point we cannot exclude the possibility that the condensation of some constructs in the wild-type background is due to the truncations or the increase in protein levels.

On the other hand, the lack of condensates in the triple deletion mutant in constructs lacking the PQ region or the coiled-coil cannot be explained by reduced protein levels. This is particularly true for 590-1381 and 1-590 variants which are present at elevated concentrations and do not form condensates. Therefore, we conclude that the diffuse localization of these constructs is due to molecular features and not concentration.

(2c) Figure 7C and l. 252+: how the behavior of the puncta formed with the Ede1(366-900)-2xPH construct depend on concentration? Also, do all the puncta recruit Sla1? Is Sla1 also recruited at sites where the Ede1 construct is not present?

The size and shape of structures formed by the Ede1(366-900)-2xPH construct depend on the concentration of the construct (Figure 9A,B; formerly 7A,B). Low expressing cells had more diffuse localization of the construct and separated into puncta or well-separated regions as the concentration increased.

Most of the puncta of Ede1 construct do recruit Sla1, but Sla1 is also recruited at sites where the Ede1 construct is not present (see kymograph in former Figure 7C, now 9F). The artificial Ede1 construct is likely less effective in recruiting the endocytic components than the natural Ede1. Thereby, allowing some sites to form independently of Ede1 as is seen in the Ede1 deletion strains.

3. l. 157: I am not sure I understand the logic (I may have missed something). The fact that it is a condensate does not imply it triggers actin polymerization. This is a correlation, not a causation. In addition, one could imagine there is a defined endocytic patch within a diffraction limited distance from the condensate. Quantifying the amount of abp1 (relatively to other patches) could be a way to tell whether these are bona fide endocytic structures coming out of the aggregate.

We did not mean to imply that actin polymerization is directly caused by the phase-separated nature of the condensates, merely that proteins present in the condensates are normally able to trigger it (e.g., Las17). We edited that line for clarity (l. 202).

4. In conclusion, in its current form, this study does convince me that Ede1 phase separates during clathrin-mediated endocytosis. In addition, it is unclear to me how the putative phase separation of Ede1 would affect endocytosis, compared to regular protein assembly. I believe several extra experiments would be necessary to make a strong point.

We thank the reviewer for their critical assessment. We hope that the new experimental data we provide in this revised manuscript now convinces the reviewer of the value of our findings. Particularly, the two key additions are: we show that the normal cytosolic Ede1 level is at the critical concentration for phase separation (new Figure 3), and that the phase separating core region of Ede1 can be functionally replaced with heterologous sequences that are known or predicted to phase separate (new Figure 8).

It is true that phase separation of diffraction-limited structures is a contentious topic that requires critical assessment (McSwiggen et al., 2019; Peng and Weber, 2019). Our new findings add to the lines of evidence already present in the original manuscript, which all point to the same phenomenon. We rewrote the discussion to put our findings in the broader context and distinguish phase separation from other types of assembly.

References

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[Editors’ note: what follows is the authors’ response to the second round of review.]

After some discussion on the new version of the manuscript, there is a general agreement on the interest of the article and the quality of the data. However, two out of three reviewers still consider that the functional relevance of liquid phase separation in endocytosis is not conclusively proven and other hypothesis might as well explain the data. Even though the observation that other unrelated Intrinsically Disorder Regions (IDRs) can partially substitute for the Ede1 regions promoting liquid phase separation is quite compelling, the inserted IDRs might as well work by providing a flexible loop of a given size between the N and C terminal domains or by promoting oligomerization. Since the role of liquid phase separation in endocytosis is under strong debate, we feel that these two other possibilities need to be properly tested and discussed before publication. Even if the new data would disprove the liquid phase separation model, the article would still be of interest for eLife. If the new oligomerization or linker regions cannot functionally substitute for the Ede1 coiled-coil (CC) and PQ rich (PQ) regions, the data would provide stronger support for the role of liquid phase separation in endocytosis but still, the title, the abstract and the discussion would need to be rephrased to avoid overstatements that would slow down advance in the field. In addition to this main concern, there is still the issue on the recruitment of the Ede1-(PQ,CC)∆ mutant to endocytic sites. At this point, it is not possible to conclude that the CC-PQ region is required for endocytic uptake because the mutant is not recruited to endocytic sites. One of the reviewers also suggested to investigate the possible differences in the posttranslational modifications that could lead to phase-separation. Even though this approach could be enlightening, it will take more than two months and it is probably beyond the scope of the manuscript. In essence then, the manuscript would be adequate for publication in eLife, provided than you address the following key points:

1. Substitute the CC and PQ regions for non-IDRs that can act as linkers or oligomerization domains and investigate if the chimeras can complement the ede1∆ phenotype.

We expanded the repertoire of protein linkers investigated in Figure 8 with four structured domains: two fluorescent proteins serving as globular linkers, and two kinesin coiled-coils. In addition, these linkers run the gamut of oligomerization states, from monomeric (mCherry), through dimeric (dTomato, kinesin-1) to tetrameric (kinesin-5).

The results suggest that Ede1 localization, but not function, can be partially rescued by an oligomerization domain. However, we saw a significant functional rescue of the late-phase patch density only when we used prion-like IDRs as replacements.

These results are consistent with the model where EH domains mediate interactions with other proteins, and the central region mediates self-interaction and condensation. Thus, increasing the valency of EH domains can rescue the recruitment of Ede1 to functional patches, but it did not rescue Ede1 function of promoting patch formation.

We would like to note that as a general principle, it is difficult to interpret this type of in vivo domain replacement experiment regardless of the result. We performed and then expanded this experiment per the reviewers’ requests, but we try to approach the results presented in Figure 8 with caution. In living cells, there are too many uncontrollable variables for us to be able to say conclusively why one replacement can rescue the defect and the others cannot. Still, these results do support our conclusion that heterologous prion-like IDRs can, at least partially, replace the function of Ede1’s core region.

2. Solve the issue of the recruitment of the Ede1-(PQ,CC)∆ to endocytic sites.

We showed previously that the deletion of the central region of Ede1 inhibits the formation of endocytic patches by the early-arriving endocytic proteins (Figure 7) and that this has downstream effects on the late-arriving Sla1 protein (Figure 6).

The reviewer suggested a possibility that the central region of Ede1 might be simply a recruitment motif, and that the terminal fragments of Ede1 could support early site formation if recruited to the membrane or endocytic proteins through other means.

We tested that possibility in the previous round of revision by artificially recruiting the Ede1 terminal domains to the early protein Syp1. This, however, did not rescue the initiation defect nor the downstream defect.

We cannot help but see this reasoning as somewhat circular: because the early patch formation requires Ede1 central region (Figure 7), we cannot recruit Ede1 which lacks the central region to early patches. We can only recruit it to other membrane-binding early endocytic proteins, which we did for Syp1, showing no rescue of Syp1 or Ede1 patch localization, and no effects downstream on the late phase proteins.

We have now repeated this experiment by recruiting the Ede1 terminal domains to Sla2 (Author response image 2). We did not feel that these data would add to the conclusions of the manuscript, so we decided to only show them here in the rebuttal.

We chose Sla2 as an anchor, because the localization of Sla2 to endocytic sites is not prevented by the Ede1 central region deletion (Figure 7). Recruiting Ede1 terminal domains to Sla2 did rescue Ede1 patch formation, although not fully (Author response image panel A). However, this did not rescue the downstream Sla1 patch density (panel B, patch density from respectively 47 and 69 cells in DMSO- and rapamycin-treated cells; line and whiskers show mean +/- SD; p-value from two-sided t-test).

As a control for the rapamycin-driven recruitment, we used a strain described by Brach et al. (2014), where three early proteins tagged with FRB are recruited to eisosomes via FKBP-tagged Pil1, and subsequently recruit Ede1-EGFP. Rapamycin treatment of this strain under the same conditions as the Sla2/Ede1∆PQCC strain resulted in increased Ede1-Pil1 colocalization compared to solvent-only treatment (panel C).

These results agree with our conclusion that the central region of Ede1 is important for its function. However, we do not feel that negative results from this kind of synthetic biology experiments are very conclusive. Alternative explanations could be that we did not recruit the correct amount of Ede1 terminal domains, or that they are not correctly localized or oriented with respect to the binding sites in the other early endocytic proteins.

In these experiments we are attempting to re-engineer Ede1, a relatively complex multidomain protein, by replacing a part of it with a heterologous sequence. These kinds of experiments are obviously very challenging, and the likelihood of failure is high. In the manuscript we conclude that the core region of Ede1 is critical for assembling Ede1 and other early endocytic proteins to endocytic sites. Furthermore, we are suggesting that the condensing ability of the core region is important for Ede1’s function. Even if we were able to engineer a variant of Ede1 that functions without self-association driven condensation, our conclusions about the function of normal Ede1 would not be affected. We never suggested that the condensing property of Ede1 would be an absolute requirement for endocytosis, nor that it could not be replaced with another mechanism. Therefore, we believe that further attempts at making Ede1 localize functionally without its natural core region are outside of the scope of this manuscript.

Author response image 2

3. Rewrite the title, the abstract and the discussion to make clear that the results support the requirement for liquid-phase separation in endocytosis but they do not conclusively prove it.

We have changed the title to use the mechanism-agnostic term Condensation in place of Phase separation. We have also made numerous changes to the text. On the whole, we consider the text to have been fairly cautious and account for possible alternative hypotheses already. Reviewer #2 notes: “(…) I think this is a careful study and the authors are quite conservative in their interpretation.

In addition to these main concerns, please carefully read the rest of the points raised by the reviewers and discuss or address them:

1. The new data showing Ede1 concentration buffering is interesting. However, we find the authors' interpretation unclear at places and, to our understanding, only one possible interpretation of the data. The authors say the cells control their cytoplasmic concentration of Ede1 but, to them, is it an active or a passive process? For bona fide purely phase separating proteins, this can be a passive process (once phase separation happens, the soluble concentration remains virtually constant and the size of the condensates changes with changing concentration). The data presented in the paper is indeed compatible with this hypothesis. However, the data is also compatible with equally likely alternative hypotheses. One is that there are two populations of Ede1 with different post-translational modifications because of the overexpression. As the authors point out, Ede1 is the most heavily phosphorylated protein in CME (Lu et al. 2016), and it is also ubiquitinated. Therefore, it is possible that overexpressing Ede1 overloads its kinases, phosphatases and/or ubiquitination machineries, and the excess mis-phosphorylated/mi-ubiquitinated proteins would phase separate, without being involved in endocytosis. It is also possible that Ede1 does not normally phase separate at endocytosis sites because it is bound to endocytic partners or cargo. Overexpressing Ede1 would create a stoichiometric imbalance at endocytic sites and the excess protein would end up in the cytoplasm and could phase separate there only.

We completely agree that condensation properties of Ede1 may be affected or regulated by phosphorylation and / or ubiquitylation. This regulation deserves investigation but is outside the scope of this article. We mention Ede1 phosphorylation in the Discussion section of the manuscript (p. 22).

On this point, we would also like to stress that one of the conclusions from the quantifications presented in Figure 3 is not merely that a threshold cytoplasmic concentration of Ede1 exists, but that wild-type cells already operate at this threshold level.

2. We find it surprising that the FUS domain does not phase separate Ede1 since FUS is a well known phase separating domain, and other phase separating domains seem to work. Do the authors have an explanation for that?

We can offer plausible speculation. FUS family proteins phase separate through a distinct mechanism mediated by tyrosine and arginine residues, and affected by phosphorylation and methylation (Qamar et al., 2018; Wang et al., 2018). The FUS low-complexity (LC) region we used readily phase separates at high concentrations in vitro, but in physiological conditions, FUS phase separation is greatly enhanced by the interactions of the LC region with the RNA recognition motif (Wang et al., 2018). Previous work in yeast has shown that FUS aggregates with cytotoxic effects when overexpressed in yeast (Fushimi et al., 2011). On the other hand, expression of LC-only constructs in yeast reduces the aggregation and cytotoxicity compared to full-length FUS (Kryndushkin et al., 2011), especially when not overexpressed. All of our constructs were expressed from Ede1 locus under the control of the native promoter, and this level may not be enough for the phase separation of the Ede1-FUS construct in yeast cells.

3. L 208: "catalyzes cycles of assembly and disassembly of actin": it is not clearly demonstrated, so this could reformulated.

We have reformulated the sentence to: ”(…) are associated with cycles of assembly and disassembly of actin”.

4. L 392: "likely": change to "possibly" because this is not demonstrated in the paper (just correlation with abp1).

We changed the phrasing.

5. In response to reviewer #2, the authors point out that hexanediol causes the PH domain to form clusters, but say they are not showing it in the paper because it is only peripherally related to the paper. I agree but this could be useful for the field to report this anyway because there are a lot of controversies about the use of hexanediol in the phase separation field.

We agree that this is an important observation, and we included it as part of Figure 9—figure supplement 1.

6. Talking about fusion and fission of condensates is an overstatement. It might just be the condensates getting closer or separating. Talking about apparent fission or fusion events throughout the text would be more accurate. How often are the putative fission events observed?

We agree with the reviewer. Therefore, the text only mentions apparent fusion and fission. From single-plane movies, we estimate that apparent fusion and fission events happen at a similar rate of about 0.0007 (cell*s)-1; this number would likely be higher if full cell volumes were considered. Apparent fusion often, but not always, follows apparent fission (i.e. a droplet splits when Abp1 is present, and subsequently reforms, as in Figure 4—video 1). We hope the reviewer finds this information useful in the interpretation and assessment of our manuscript.

7. Is the PQ region alone generating condensates? The authors test the 1-591 Ede1 region containing the EH domains and the PQ region, but not the PQ region alone. The EH domain might somehow inhibit the formation of condensates by the IDR.

We have not tried this. It would be an interesting additional experiment, but we believe it is outside the scope of this current manuscript.

8. Are the condensates co-localizing FM4-64 stainable membranes? If so, the membrane association might provide for the liquid-phase-like behavior.

We performed this experiment. The condensates and membranes stained by FM4-64 do not show any apparent colocalization (Figure 4—figure supplement 1).

9. Does Abp1 assemble on all condensates on the time-lapse movies? Does the transient Abp1 assembly on the condensates explain the low percentage of co-localization? Are condensates hot spots for Abp1 assembly?

Not all condensates recruit Abp1 in our movies, but this is unsurprising given the typical duration of ~2 minutes per movie. The transient assembly seen in the movies is the simplest and most obvious explanation for the low co-localization rates, which were measured by analyzing images of single timepoints. Abp1 can assemble on the same condensates multiple times.

10. Are the GFP-Ede1(366-900) condensates behaving as the GFP-Ede1 condensates in terms of temperature and hexanodiol sensitivity? It is surprising that the GFP- Ede1(366-900)-2xPH condensates do not. If the GFP-Ede1(366-900) condensates behave as separate liquid-phase condensates, similar to those generated by Ede1, it might mean that their membrane association modifies the Ede1-liquid-phase properties. In this context, are the PM associated Ede1 condensates (dome-shaped) behaving similar to those assembled within the cytosol?

We present a qualitative comparison of hexanediol effects on GFP-Ede1(366-900) and GFP- Ede1(366-900)-2xPH in Figure 9—figure supplement 1. In short, the membrane-bound construct is not affected by hexanediol treatment. The GFP-Ede1(366-900) condensates are mostly or completely dissolved by hexanediol (there is some cell-to-cell variability; figure shows the typical scenario).

11. It is still not so clear if the GFP- Ede1(366-900)-2xPH can really initiate bona fide endocytic events. The internalization of the Sla1 patches is not clear in the images provided. If they are bona fide endocytic events, the fact that GFP- Ede1(366-900)-2xPH condensates do not behave as a liquid-phase would suggest that the formation of an Ede1-dependent liquid phase is not really essential for endocytic uptake and that the Ede1 central region rather operates as a linker or by recruiting other endocytic proteins.

First, we want to emphasize that we have never claimed that Ede1 condensation in yeast is essential for endocytic uptake. Rather we are suggesting that Ede1 condensation promotes endocytosis by increasing the initiation rate and concentrating cargo-specific adaptors. Ede1 itself is not required for endocytosis. In fact, none of the early-arriving proteins, including clathrin and the AP2 complex, are individually required for endocytic initiation in yeast. We would like to reiterate that the assembly of the endocytic sites is mechanistically flexible and promoted by many different interactions; this point is made repeatedly in the Discussion section of our manuscript.

The take-away of Figure 9 is that the central region of Ede1 can cause clustering of proteins at the plasma membrane. We included the observation that Sla1 also assembles on GFP- Ede1(366-900)-2xPH clusters because we believe it could be interesting to the readers, even if we cannot fully explain it in the present manuscript. Discussion (p.22): “It must be noted that we do not fully understand the nature of the microdomains formed by the GFP-Ede1-PQCC-2×PH construct. For example, all known interaction motifs of Ede1 are located inside the terminal regions. It is thus unclear how the central region could recruit other endocytic proteins.” We have deleted the references to this observation from the abstract and the introduction.

12. The Rapamycin inducible system might not be the most adequate to bring the Ede1 trucations to endocytic sites because the tor1 mutations used to make the yeast strain resistant to Rapamycin might interfere with endocytic uptake. Are Syp1 and Sla1 cortical patch dynamics similar to the wild type´s in a tor1-1 frp1 EDE1-mCherry-FKPB SYP1-FRB background? It might be easier to covalently link the Ede1 truncations to Syp1 or to a later endocytic coat component that is less affected by depletion of Ede1.

The tor1-1 strains have been used previously to study endocytosis and no obvious interference has been observed (e.g. Brach et al., 2014). We agree that there might be other more optimal ways to perform this experiment that was suggested by the reviewer. However, it is very challenging to engineer synthetic protein constructs that could functionally replace normal endocytic proteins.

We re-emphasise the point that we are not claiming that Ede1’s condensation activity is required for endocytic initiation. Rather, we are suggesting that Ede1’s condensation activity contributes to initiation in wild type cells. Therefore, even if we managed to generate a fully functional version of Ede1 that bypasses the need for the natural core region, it would not mean that in normal cells Ede1’s core is not promoting endocytic initiation.

Reviewer #1:

Kozak and Kaksonen demonstrate that the mammalian Eps15 yeast homolog Ede1 can phase-separate upon overexpression or deletion of endocytic adaptors in vivo. The authors define the protein regions necessary and sufficient to generate the condensates and demonstrate that those are probably necessary and sufficient to sustain the endocytic function of Ede1. Conclusive demonstration on whether liquid phase-separation actually occurs at endocytic sites in wild type cells remains elusive. However, the authors show that unrelated protein sequences known to undergo this transition can functionally (albeit partially) substitute for the Ede1 region generating the condensates.

Even though the data points to the functional role of a separated Ede1 liquid phase in endocytic uptake, some important issues are still unsolved:

1. The authors conclude that the coiled-coil and IDR of Ede1 is required for endocytic uptake because using a Rapamycin inducible system to hook the Ede1 mutant lacking these domains to Syp1, does not complement the ede1∆ defects. Again, this particular experiment is not very informative because the Ede1 mutant is not really recruited to endocytic sites in the presence of Rapamycin and therefore, the endocytic defect might be because by the absence of the N or C-terminal domains.

Strictly speaking, neither Ede1 nor any other early-arriving protein, including clathrin, is required for endocytic uptake. We do not claim that they are, and we do propose that multiple mechanisms, including condensation mediated by Ede1, contribute to the assembly of the endocytic coat and the concentration of cargo.

We elaborated on this experiment in the response to the editor. We repeated this experiment by recruiting the Ede1 terminal domains to Sla2. We were successful in partially re-establishing Ede1 patch formation but did not see a change in Sla1 patch density upon rapamycin treatment.

Perhaps the fusion constructs presented in Figure 8 will provide some evidence of the kind requested by the reviewer: the dimeric constructs partially rescue localization but fail to rescue the late-phase site density.

The Rapamycin inducible system might not be the most adequate to bring the Ede1 trucations to endocytic sites because the tor1 mutations used to make the yeast strain resistant to Rapamycin might interfere with endocytic uptake. Are Syp1 and Sla1 cortical patch dynamics similar to the wild type´s in a tor1-1 frp1 EDE1-mCherry-FKPB SYP1-FRB background? It might be easier to covalently link the Ede1 truncations to Syp1.

Other important but easily addressable points:

1. Talking about fusion and fission of condensates is an overstatement. It might just be the condensates getting closer or separating. Talking about apparent fission or fusion events throughout the text would be more accurate. How often are the putative fission events observed?

2. Is the PQ region alone generating condensates? The authors test the 1-591 Ede1 region containing the EH domains and the PQ region, but not the PQ region alone. The EH domain might somehow inhibit the formation of condensates by the IDR.

3. Are the condensates co-localizing FM4-64 stainable membranes? If so, the membrane association might provide for the liquid-phase-like behavior.

4. Does Abp1 assemble on all condensates on the time-lapse movies? Does the transient Abp1 assembly on the condensates explain the low percentage of co-localization? Are condensates hot spots for Abp1 assembly?

5. Are the GFP-Ede1366-900 condensates behaving as the GFP-Ede1 condensates in terms of temperature and hexanodiol sensitivity? It is surprising that the GFP- Ede1366-900-2xPH condensates do not. If the GFP-Ede1366-900 condensates behave as separate liquid-phase condensates, similar to those generated by Ede1, it might mean that their membrane association modifies the Ede1-liquid-phase properties. In this context, are the PM associated Ede1 condensates (dome-shaped) behaving similar to those assembled within the cytosol?

6. It is still not so clear if the GFP- Ede1366-900-2xPH can really initiate bona fide endocytic events. The internalization of the Sla1 patches is not clear in the images provided. If they are bona fide endocytic events, the fact that GFP- Ede1366-900-2xPH condensates do not behave as a liquid-phase would suggest that the formation of an Ede1-dependent liquid phase is not really essential for endocytic uptake and that the Ede1 central region rather operates as a linker or by recruiting other endocytic proteins.

We have addressed the reviewer's minor points 1-6 in the response to the editor’s summary.

Reviewer #3:

We thank the authors for their efforts in addressing our initial concerns and providing new data. We think the paper has been improved since the first submission. However, we think there are still important remaining issues.

1. The authors provide a more thorough characterization of the phase separated Ede1 structures observed in mutant cells or after over-expression. However, we are still not completely convinced phase separation does happen during endocytosis and is functionally important for this process. We agree with the authors that all the data are "compatible" with this idea, but they are also compatible with the idea that phase separation is a protection mechanism for excess protein or non-functional proteins. The authors acknowledge that in the response to reviewers ("we cannot exclude the possibility that the condensation of some constructs in the wild-type background is due to the truncations or the increase in protein levels") and use "likely" very often in their response when talking about phase separation during endocytosis. Most arguments we used in our first review are still valid even with the new data. We acknowledge that completely disproving the alternative hypotheses will be tedious but at least the text should present more clearly that several interpretations are compatible with the data, and the title and abstract should be toned down.

We made several changes to the abstract, as well as changed ‘phase separation’ to ‘condensation’ in the title. We hope that the Discussion section in its current state considers various caveats and alternative mechanisms to the reviewer’s satisfaction.

2. The new data showing Ede1 concentration buffering is interesting. However, we find the authors' interpretation unclear at places and, to our understanding, only one possible interpretation of the data. The authors say the cells control their cytoplasmic concentration of Ede1 but, to them, is it an active or a passive process? For bona fide purely phase separating proteins, this can be a passive process (once phase separation happens the soluble concentration remains virtually constant and the size of the condensates changes with changing concentration). The data presented in the paper is indeed compatible with this hypothesis. However, the data is also compatible with equally likely alternative hypotheses. One is that there are two populations of Ede1 with different post-translational modifications because of the overexpression. As the authors point out, Ede1 is the most heavily phosphorylated protein in CME (Lu et al. 2016), and it is also ubiquitinated. Therefore, it is possible that overexpressing Ede1 overloads its kinases, phosphatases and/or ubiquitination machineries, and the excess mis-phosphorylated/mi-ubiquitinated proteins would phase separate, without being involved in endocytosis. It is also possible that Ede1 does not normally phase separate at endocytosis sites because it is bound to endocytic partners or cargo. Overexpressing Ede1 would create a stoichiometric imbalance at endocytic sites and the excess protein would end up in the cytoplasm and could phase separate there only.

3. The new data where the PQCC domains are replaced with known phase separating domains is nice and interesting. We are wondering if phase separation is required or if strong dimerization or oligomerization would be sufficient for the rescue? This hypothesis is compatible with the fact that the effects of the ΔCC and ΔPQ deletions seem additive and that the ΔCC (possibly a dimerization domain) mutant has very weak localization to endocytic patches. To test this hypothesis, the authors could try to rescue the ΔCC, ΔPQ and ΔPQCC mutants with dimerization or oligomerization domains, instead of phase separating domains.

We find reviewer’s points 2-3 useful and have addressed them in the response to the editor’s summary.

https://doi.org/10.7554/eLife.72865.sa2

Article and author information

Author details

  1. Mateusz Kozak

    Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Geneva, Switzerland
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1354-693X
  2. Marko Kaksonen

    Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Geneva, Switzerland
    Contribution
    Conceptualization, Funding acquisition, Writing - original draft, Writing - review and editing
    For correspondence
    marko.kaksonen@unige.ch
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3645-7689

Funding

Swiss National Science Foundation (31003A_163267)

  • Marko Kaksonen

Swiss National Science Foundation (310030B_182825)

  • Marko Kaksonen

NCCR Chemical Biology

  • Marko Kaksonen

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

Acknowledgements

This work was supported by the Swiss National Science Foundation (grants 31003A_163267 and 310030B_182825) and by the NCCR Chemical Biology funded by the SNSF.

We are thankful to all the members of the Kaksonen laboratory, especially Markus Mund, Andrea Picco, and Daniel Hummel for their critical reading of the manuscript. We also thank Jeanne Stachowiak and Kasey Day for their comments and Camilla Godlee for contributions to the early phase of the project.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. María Isabel Geli, Institut de Biología Molecular de Barcelona (IBMB), Spain

Reviewer

  1. Stephen J Royle, Warwick Medical School, United Kingdom

Publication history

  1. Preprint posted: December 2, 2019 (view preprint)
  2. Received: August 6, 2021
  3. Accepted: April 1, 2022
  4. Accepted Manuscript published: April 12, 2022 (version 1)
  5. Version of Record published: May 3, 2022 (version 2)

Copyright

© 2022, Kozak and Kaksonen

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.

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  1. Mateusz Kozak
  2. Marko Kaksonen
(2022)
Condensation of Ede1 promotes the initiation of endocytosis
eLife 11:e72865.
https://doi.org/10.7554/eLife.72865

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