Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

  1. Maia Kinnebrew
  2. Giovanni Luchetti
  3. Ria Sircar
  4. Sara Frigui
  5. Lucrezia Vittoria Viti
  6. Tomoki Naito
  7. Francis Beckert
  8. Yasunori Saheki
  9. Christian Siebold
  10. Arun Radhakrishnan
  11. Rajat Rohatgi  Is a corresponding author
  1. Department of Biochemistry and Medicine, Stanford University School of Medicine, United States
  2. Department of Physiological Chemistry, Genentech, United States
  3. Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, United Kingdom
  4. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
  5. Department of Molecular Genetics, University of Texas Southwestern Medical Center, United States

Abstract

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

Editor's evaluation

This paper addresses an important question regarding the mechanisms of Hedgehog signaling. The authors develop a new method to observe changes in cholesterol accessibility in the outer lamella of the plasma membrane to investigate the activity of the Hedgehog receptor PTCH1 and its modulation by Sonic Hedgehog. The results support the conclusion that PTCH1 needs a potassium gradient to reduce chemically-active cholesterol in the outer lamella, presumably by translocating the sterol to the inner lamella. The proposed model contradicts previous reports that suggest transport in the opposite direction using the plasma membrane sodium gradient for energy. In the initial review, the reviewers appreciated the potential impact of the findings and suggested several areas for improvement. The authors have now satisfactorily addressed the reviewers' comments in the revised manuscript.

https://doi.org/10.7554/eLife.70504.sa0

Introduction

Patched 1 (PTCH1) is a 12-pass transmembrane (TM) receptor for Hedgehog (Hh) ligands that was cloned over 30 years ago in Drosophila (Hooper and Scott, 1989; Nakano et al., 1989). PTCH1 keeps Hh signaling in the off state by inhibiting the function of Smoothened (SMO), a member of the Frizzled-class G protein-coupled receptor family (reviewed in Kong et al., 2019). Hh ligands like Sonic Hedgehog (SHH) bind and inactivate PTCH1, allowing SMO to adopt an active conformation and transduce the Hh signal across the membrane. Inactivating mutations in PTCH1 lead to unrestrained Hh signaling and cause birth defects and human cancers. Despite three decades of research, the biochemical activity of PTCH1, and how this activity inhibits SMO, has remained mysterious.

An early clue to the function of PTCH1 (confirmed more recently by structural studies) was the observation that its sequence was similar to Niemann-Pick C1 (NPC1), a protein that transports cholesterol from the lumen of the lysosome to the cytoplasm (Carstea et al., 1997; Gong et al., 2018; Gong et al., 2016; Li et al., 2016; Loftus et al., 1997; Qian et al., 2020; Qi et al., 2018a; Qi et al., 2018b; Zhang et al., 2018). PTCH1 overexpression was also shown to enhance the efflux of a fluorescent analog of cholesterol (BODIPY-cholesterol) from cells (Bidet et al., 2011). A second clue was the observation that both oxysterols (Nachtergaele et al., 2012) and cholesterol (Byrne et al., 2016; Huang et al., 2016; Luchetti et al., 2016) directly bind SMO and were sufficient to activate Hh signaling (even in the absence of Hh ligands). These latter studies led to the model that PTCH1 uses its transporter function to somehow prevent cholesterol from binding and activating SMO (Kong et al., 2019; Kowatsch et al., 2019).

To identify the second messenger that communicates the signal between PTCH1 and SMO, we previously conducted an unbiased CRISPR screen to find cellular lipids that influence Hh signaling (Kinnebrew et al., 2019). The screen identified multiple genes encoding enzymes at all levels in the cholesterol biosynthesis pathway as positive regulators, confirming the central importance of cholesterol in Hh signaling. More importantly, it implicated a minor pool of membrane cholesterol, termed accessible cholesterol, in the communication between PTCH1 and SMO. A large body of data (summarized in Das et al., 2014; Lange and Steck, 2020; McConnell and Radhakrishnan, 2003) supports the model that cholesterol in the plasma membrane segregates into at least two pools. The major pool of plasma membrane cholesterol is sequestered in complexes with phospholipids, particularly sphingomyelin (SM), and is inaccessible to proteins or soluble acceptors. In contrast, a minor pool of plasma membrane cholesterol is free from interactions with other lipids and is accessible to modulate the activity of proteins or to escape the membrane to soluble acceptors. The major physicochemical difference between cholesterol in these two pools is its chemical activity (a quantity related to chemical potential), which is reduced by the formation of complexes with membrane phospholipids. Selectively changing accessible cholesterol (without altering total cholesterol) in membranes altered Hh signaling strength in a predictable manner: increasing accessible cholesterol by depleting SM potentiated Hh signaling while trapping accessible cholesterol using a bacterial toxin domain (ALOD4) attenuated Hh signaling (Kinnebrew et al., 2019; Radhakrishnan et al., 2020).

These studies led us to propose the model that PTCH1 depletes accessible cholesterol to block SMO activity (Kinnebrew et al., 2019; Radhakrishnan et al., 2020). To test this hypothesis, we established a direct assay for the biochemical activity of PTCH1 and used it to investigate the transport mechanism.

Results

Conflicting models for the influence of PTCH1 on membrane cholesterol organization

Previous studies have evaluated the effect of PTCH1 on membrane cholesterol by measuring the steady-state binding of fluorescent cholesterol sensors derived from microbial toxins like Perfringolysin O (PFO). Using a fluorescently labeled, non-lytic version of PFO called PFO*, we found that PTCH1 inactivation by SHH leads to increased accessible cholesterol in the outer leaflet of the membrane surrounding primary cilia, the cellular compartment where SMO triggers downstream signaling (Kinnebrew et al., 2019). Studies from other groups using PTCH1 expressed throughout the plasma membrane concluded that PTCH1 inactivation increases cholesterol levels in the inner leaflet (Zhang et al., 2018). This study used a probe derived from the cholesterol-binding D4 domain of PFO (PFOD4) carrying several mutations and modifications that increased its cholesterol sensitivity (Liu et al., 2017). One important difference between the two studies is that the engineered PFOD4 probes used in the latter study are not selective for accessible cholesterol, binding to cholesterol regardless of the surrounding phospholipid environment (Liu et al., 2017).

To measure inner leaflet accessible cholesterol in membranes of live cells with or without PTCH1, we used a version of PFOD4 containing a single point mutation (D434S), called PFOD4H, that retains selectivity for accessible cholesterol (Abe and Kobayashi, 2021; Gay et al., 2015; Johnson et al., 2012; Maekawa and Fairn, 2015). Membrane recruitment of cytoplasmically expressed PFOD4H has been shown to directly reflect the abundance of inner leaflet cholesterol (Maekawa and Fairn, 2015). Using a plasmid that fused the coding sequence of PFOD4H to the TdTomato fluorescent protein, we expressed TdTomato-PFOD4H in the cytoplasm of HEK293T cells and monitored its recruitment to the inner leaflet of the plasma membrane by total internal reflection fluorescence microscopy (TIRFM) (Maekawa and Fairn, 2015). The limited axial depth of fluorophore excitation by TIRFM allowed us to readily measure the amount of TdTomato-PFOD4H bound to the plasma membrane in live cells, without interference from the TdTomato-PFOD4H in the cytoplasm or bound to intracellular organelles (Figure 1A). To evaluate the effect of PTCH1, we used HEK293T cells stably expressing PTCH1 under the control of a Doxycycline (Dox)-inducible promoter. PTCH1 expression increased TdTomato-PFOD4H binding to the inner leaflet of the plasma membrane in a manner that could be blocked by its inhibitory ligand SHH (Figure 1B), consistent with the model that PTCH1 increases inner leaflet cholesterol accessibility.

Figure 1 with 1 supplement see all
PTCH1 activity increases inner leaflet cholesterol.

(A) Total internal reflection fluorescence microscopy (TIRFM) was used to measure the recruitment of two different fluorescent cholesterol sensors (TdTomato-PFOD4H or GFP-GRAM1b) to the inner leaflet of the basal plasma membrane adjoining the coverslip. TIRFM only excites the sensor molecules (dark green) present in the ~100-nm-wide illumination zone, thus excluding fluorescence from the majority of sensor molecules present in the cytoplasm or bound to internal organelles (light green). (B, C) Steady-state TdTomato-PFOD4H (B) or GFP-GRAM1b (C) fluorescence at the inner leaflet of the plasma membrane in HEK293T cells with or without PTCH1 expression induced by the addition of Doxycycline (Dox). SHH (1 μM) was used to inactivate PTCH1 where indicated. Each circle represents the average intensity of fluorescence from a single cell membrane (n>65 cells), with the horizontal line representing the median and the vertical line denoting the interquartile range. Statistical significance was determined with a Mann-Whitney test. p-values in (B) are: −Dox vs. +Dox<0.0001, +Dox vs. +Dox + SHH<0.0001 and −Dox vs. +Dox + SHH=0.0127. p-values in (C) are: −Dox vs. +Dox<0.0001, +Dox vs. +Dox + SHH<0.0001 and −Dox vs. +Dox + SHH=0.0002. Experiments in (B) and (C) were repeated three independent times with similar results.

Since these results were different from those reported previously (Zhang et al., 2018), we repeated these measurements using a eukaryotic cholesterol-binding domain, known as a GRAM domain, from the GRAMD1b protein fused to GFP (GFP-GRAM1b). GFP-tagged GRAM domains expressed in cells have recently been used to measure changes in inner leaflet cholesterol in response to cholesterol loading or extraction using TIRFM (Ercan et al., 2021; Naito et al., 2019). GRAM domains bind to excess accessible cholesterol in the inner leaflet of the plasma membrane in conjunction with phosphatidylserine, allowing the GRAMD1 proteins to transport accessible cholesterol from the plasma membrane to the endoplasmic reticulum (ER) at ER-plasma membrane contact sites (Ercan et al., 2021; Ferrari et al., 2020; Naito et al., 2019; Naito and Saheki, 2021; Sandhu et al., 2018). PFOD4H and GRAM1b have no structural similarity to each other, with the former derived from a bacterial toxin and the latter from a eukaryotic protein. Steady-state binding measurements of GFP-GRAM1b again supported the model that PTCH1 increases inner leaflet cholesterol accessibility (Figure 1C).

To measure outer leaflet cholesterol accessibility in these same cells, we used a fluorescently labeled cholesterol-binding domain (mNeon-ALOD4) from a different microbial toxin called Anthrolysin O (ALO) (Endapally et al., 2019; Johnson and Radhakrishnan, 2021). We observed small but consistent changes in outer leaflet cholesterol that were complementary to those seen at the inner leaflet: PTCH1 activity decreased outer leaflet cholesterol accessibility (Figure 1—figure supplement 1). These results are in agreement with our previous observations that PTCH1 decreases cholesterol accessibility in the outer leaflet of the ciliary membrane of fixed cells (Kinnebrew et al., 2019).

Given the apparently conflicting results using steady-state binding of probes based on pore-forming toxins like PFO or ALO and the caveats associated with these probes (noted previously by independent investigators [Courtney et al., 2018]), we sought to measure the effect of PTCH1 on membrane cholesterol by developing a completely different assay.

A kinetic assay for accessible cholesterol in membranes

To test the hypothesis that PTCH1 can reduce accessible cholesterol in membranes of live cells, we first established an assay to measure this pool of cholesterol. Methyl-β-cyclodextrin (MβCD) is a cyclic oligosaccharide with a polar surface and a hydrophobic cavity that can rapidly extract cholesterol from both model and cellular membranes (Christian et al., 1997; Zidovetzki and Levitan, 2007). MβCD is a useful probe for the organization of cholesterol in the membrane because it bypasses the rate-limiting step in cholesterol extraction, the energetically unfavorable transfer of cholesterol from the membrane to the adjacent aqueous layer (Kilsdonk et al., 1995). MβCD is also non-intrusive because it does not destabilize lipid-lipid interactions or change membrane surface properties (Ohvo and Slotte, 1996).

A large body of experimental and theoretical work on defined lipid monolayers and bilayers has shown that the rate constant for cholesterol removal by MβCD is proportional to the chemical activity or accessibility of cholesterol (Lange et al., 2004; Litz et al., 2016; McConnell and Radhakrishnan, 2003; Ohvo and Slotte, 1996; Radhakrishnan and McConnell, 2000). For instance, SM, the phospholipid that forms the most stable complexes with cholesterol, markedly reduces the rate of cholesterol removal by MβCD from lipid monolayers (Ohvo and Slotte, 1996). MβCD (which is membrane impermeable) also extracts cholesterol from intact cells when added to the extracellular medium by accepting cholesterol that desorbs from the outer leaflet of the plasma membrane. Interestingly, MβCD-catalyzed cholesterol efflux from cells follows biexponential kinetics, suggesting the presence of two kinetic pools of cholesterol: a fast pool with a t1/2 of <1 min and a slow pool with a t1/2 of >10 min (Yancey et al., 1996). The fast pool represents the accessible cholesterol pool because its size can be expanded by depleting SM and other phospholipids that sequester cholesterol (Haynes et al., 2000).

Since cholesterol can exchange rapidly between the two leaflets of the plasma membrane, removal of cholesterol from the outer leaflet with MβCD should also deplete cholesterol from the inner leaflet (Figure 2A). Consequently, we reasoned that the decrease in inner leaflet cholesterol could be used to monitor the extraction of cholesterol from the outer leaflet by MβCD. To measure changes in inner leaflet cholesterol of live cells, we used TIRFM to measure membrane recruitment of cytoplasmically expressed TdTomato-PFOD4H (Figure 2A, B). Addition of MβCD to the extracellular medium led to a decrease in TdTomato-PFOD4H fluorescence at the plasma membrane and, concomitantly, an increase in TdTomato-PFOD4H fluorescence in the cytoplasm, consistent with recent observations in HeLa cells (Abe and Kobayashi, 2021; Figure 2C).

Figure 2 with 1 supplement see all
Measurement of outer leaflet accessible cholesterol in live cells with total internal reflection fluorescence microscopy (TIRFM).

(A) Extraction of outer leaflet accessible cholesterol by MβCD results in detachment of TdTomato-PFOD4H from the inner leaflet of the plasma membrane due to flip-flop of cholesterol between the two leaflets. Outer leaflet cholesterol can exchange between two pools: accessible cholesterol (dark blue) or sequestered cholesterol (light blue). Sphingomyelin (dark gray), present exclusively in the outer leaflet, plays a dominant role in sequestering cholesterol. Accessible cholesterol (drawn as projecting further out of the surface of the membrane into the aqueous boundary layer) has higher chemical activity and thus greater propensity to interact with proteins or to be transferred to a soluble acceptor like MβCD. (B) Schematic of the assay to detect outer leaflet accessible cholesterol using TIRFM. TdTomato-PFOD4H (green ovals) expressed intracellularly in HEK293T cells is recruited to the inner leaflet of the basal plasma membrane by binding to cholesterol (see (A)), bringing it into the TIRF illumination zone. MβCD added to the media (right) extracts outer leaflet accessible cholesterol, resulting in depletion of inner leaflet cholesterol and detachment of TdTomato-PFOD4H from the membrane. (C) Images of TdTomato-PFOD4H at the membrane (top panel, TIRFM) or in the cytoplasm (bottom panel, epifluorescence microscopy) of a single cell after MβCD addition (316 μM). Dotted lines mark the cell border (top row) or nuclear border (bottom row). Scale bar is 5 µm. (D) Time course of the change in TdTomato-PFOD4H membrane and cytoplasmic fluorescence (measured every 2 s by TIRFM or epifluorescence microscopy, respectively) after MβCD addition (316 μM) (quantified from images of the type shown in (C)). Black arrows mark the time point in the experiment when MβCD was added. Each curve represents the mean fluorescence from six or more cells, with standard error of the mean (SEM) shown in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data (left y-axis) and relative to the ending fluorescence (t/t300) for the epifluorescence microscopy data (right y-axis). This experiment was repeated five independent times with similar results. (E, F) Time course of the change in TdTomato-PFOD4H membrane fluorescence measured by TIRFM after MβCD addition (316 μM, arrow) in control cells or in cells subjected to two treatments. In (E), cells were pre-incubated with ALOD4 or an ALOD4 mutant defective in cholesterol binding (added at 5 μM for 45 min prior to the assay). In (F), cells were treated with 80 μM myriocin for 3 days to deplete cellular SM. Each curve shows the mean fluorescence measured from >20 cells taken from at least three biological replicates, with SEM depicted in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data. Curves showing data without normalization to baseline fluorescence values are provided in Figure 2—figure supplement 1C, D. In (E) and (F), curve fits (see Materials and methods) were used to determine the time required for TdTomato-PFOD4H fluorescence at the plasma membrane to drop by one-half of its starting value (t1/2), with the 95% confidence interval (CI) shown in parentheses. The experiment was repeated three independent times with similar results.

Figure 3 with 2 supplements see all
PTCH1 depletes outer leaflet accessible cholesterol.

(A) Cartoon representations of PTCH1 bound to SHH and PTCH1-ᐃL2. Deletion of the second extracellular loop (L2) in PTCH1-ᐃL2 abolishes both SHH binding and a putative tunnel through PTCH1 that has been proposed to form a conduit for cholesterol transport. (B) Immunoblot showing Doxycycline (Dox)-inducible expression of stably expressed PTCH1 and PTCH1-ᐃL2 in HEK293T cells. (C) Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in the presence (+Dox) or absence (−Dox) of PTCH1 expression. SHH is added (1 μM, red curve only) during imaging to inactivate PTCH1. (D, E) The experiment in (C) was performed at various concentrations of MβCD either without (D, −Dox) or with (E, +Dox) PTCH1 expression. (F) The maximum rate of change in TdTomato-PFOD4H membrane fluorescence (dF/dt) is depicted as a function of MβCD concentration. At each MβCD concentration, the max dF/dt was calculated from the curves shown in (D) and (E). (G) The maximum rate of cholesterol extraction (dF/dt) by MβCD (316 μM) was measured at increasing concentrations of SHH in cells expressing PTCH1. The individual curves used to measure the max dF/dt values are shown in Figure 3—figure supplement 1B. (H) Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in PTCH1-expressing cells (+Dox) with or without SHH addition (1 μM, red and pink curves only). Cells were treated with either ALOD4 or an ALOD4 mutant defective in cholesterol binding (added at 5 μM for 45 min prior to assay). (I) Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in the presence (+Dox) or absence (−Dox) of PTCH1-ᐃL2 expression. Red curve depicts the effect of SHH addition (1 μM, arrow) in PTCH1-ᐃL2 expressing cells. For (C–E), (H), and (I), each curve shows the mean fluorescence measured from >20 cells taken from at least three biological replicates, with SEM depicted in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data. For (C) and (I), curves showing data without normalization to baseline fluorescence values are provided in Figure 3—figure supplement 2A B. The t1/2, along with the 95% CI, is shown for each curve. All experiments were repeated three independent times, with the exception of (D) and (E) which were performed two times. CI, confidence interval; SEM, standard error of the mean.

Figure 3—source data 1

Dotted lines mark the cropped region of the immunoblot that was used to generate panel Figure 3B.

As denoted on the left immunoblot PTCH1 (ptc) samples were run in duplicate.

https://cdn.elifesciences.org/articles/70504/elife-70504-fig3-data1-v2.pdf

Instead of using the absolute steady-state values of TdTomato-PFOD4H fluorescence at the basal membrane, we used the kinetics of extraction as a measure of cholesterol accessibility. Kinetic assays are generally more sensitive and less susceptible to artifacts. For example, the steady-state binding of TdTomato-PFOD4H to the inner leaflet can be influenced by many factors other than the cholesterol content, such as the amount of TdTomato-PFOD4H expressed in an individual cell and the non-specific affinity of the probe for membranes (Courtney et al., 2018). In contrast, cholesterol extraction by MβCD follows first-order, exponential decay kinetics (Figure 2D): the half-life (t1/2) for extraction is independent of the starting baseline value (as it is for radioactive decay). To reliably measure the kinetics of cholesterol extraction under each condition, we combined results from multiple individual cells. Each of these cells shows a different baseline level of TdTomato-PFOD4 fluorescence at the membrane, likely because each cell expresses different amounts of the probe (see the wide distribution of baseline values in Figure 1B). To focus on the kinetics (or t1/2 of extraction) we normalized the fluorescence for each cell to its own baseline value (at time=0) before averaging over all the cells analyzed. Extraction curves without baseline normalization are shown in the figure supplements for selected panels in Figures 2 and 3.

After addition of MβCD, the t1/2 of TdTomato-PFOD4H detachment was <1 min (Figure 2D), consistent with the rapid kinetics of accessible cholesterol depletion by MβCD observed in prior studies using both defined lipid monolayers and cells (Haynes et al., 2000; Ohvo and Slotte, 1996; Radhakrishnan and McConnell, 2000; Yancey et al., 1996). The detachment of TdTomato-PFOD4H from the inner leaflet of the plasma membrane and its release into the cytoplasm indicates that removal of cholesterol by MβCD from the outer leaflet also causes the depletion of cholesterol from the inner leaflet, confirming the rapid exchange or flip-flop of cholesterol between the two leaflets (Figure 2A).

To test if the kinetics of MβCD-induced TdTomato-PFOD4H detachment was influenced by the size of the outer leaflet accessible cholesterol pool, we increased or decreased membrane accessible cholesterol using two chemically distinct perturbations. First, the cholesterol-binding D4 domain of the bacterial toxin Anthrolysin O (ALOD4) was added to the extracellular medium to trap outer leaflet accessible cholesterol. This protein binds specifically to accessible cholesterol and sequesters it without altering the total cholesterol levels in the membrane (Infante and Radhakrishnan, 2017). Consistent with its cholesterol-sequestering properties, wild-type (WT) ALOD4 (but not a mutant that cannot bind cholesterol) nearly abolished MβCD-induced detachment of TdTomato-PFOD4H from the membrane (Figure 2E). We observed that ALOD4 caused a decrease in the steady-state TdTomato-PFOD4H binding to the inner leaflet, likely because sequestration of accessible cholesterol in the outer leaflet drove the movement of cholesterol from the inner to the outer leaflet by mass action (Figure 2—figure supplement 1A).

To increase the pool of outer leaflet accessible cholesterol, we depleted cells of SM, a lipid exclusively present in the outer leaflet of the plasma membrane that plays a dominant role in sequestering accessible cholesterol. SM depletion liberates accessible cholesterol without altering total cholesterol in the plasma membrane (Das et al., 2014; Tafesse et al., 2015). Myriocin, a drug commonly used to deplete SM, increased the steady-state binding of TdTomato-PFOD4H to the inner leaflet, likely because some of the accessible cholesterol liberated from the outer leaflet moved to the inner leaflet (again showing free exchange of accessible cholesterol between the two leaflets) (Figure 2—figure supplement 1B). Depletion of SM both accelerated the rate of cholesterol extraction by MβCD and increased the total amount of cholesterol extracted, consistent with expansion of the outer leaflet pool of accessible cholesterol (Figure 2F). Taken together, our TIRFM assay shows that the kinetics of MβCD-induced detachment of TdTomato-PFOD4H in live cells can be used to measure the pool of accessible cholesterol in the outer leaflet of the plasma membrane. Although TdTomato-PFOD4H is bound to the inner leaflet and MβCD extracts cholesterol from the outer leaflet (Figure 2A), the rapid exchange of cholesterol between the two leaflets allows detachment of the TdTomato-PFOD4H from the inner leaflet to reflect cholesterol extraction from the outer leaflet.

PTCH1 reduces accessible cholesterol in the membrane outer leaflet

Armed with this cholesterol transport assay, we directly assessed the effects of PTCH1 on accessible cholesterol in HEK293T cells expressing PTCH1 under the control of a Dox-inducible promoter. Control cell lines expressed a widely used truncation mutant of PTCH1 that lacks its second extracellular loop (hereafter called PTCH1-ᐃL2) under control of the same Dox-inducible promoter (Figure 3A and B). PTCH1-ᐃL2 inhibits SMO activity but cannot bind SHH and thus cannot be inactivated (Briscoe et al., 2001; Marigo et al., 1996).

Expression of PTCH1 induced by Dox addition prevented MβCD-induced detachment of TdTomato-PFOD4H from the inner leaflet (Figure 3C). PTCH1 induction did not change the abundance of TdTomato-PFOD4H in cells (Figure 3—figure supplement 1A). Measuring extraction kinetics at a series of MβCD concentrations provided a quantitative assessment of the effect of PTCH1. Without PTCH1 induction, MβCD concentrations as low as 80 μM led to TdTomato-PFOD4H detachment from the membrane; in contrast, in the presence of PTCH1, cells were resistant to MβCD even when concentrations exceeded 300 μM (Figure 3D, E). The concentration of MβCD required to achieve the half-maximum detachment rate was increased from ~170 μM to ~700 μM when PTCH1 was expressed in the plasma membrane (Figure 3F). PTCH1 expression does not completely prevent MβCD from removing membrane cholesterol; it simply shifts the MβCD concentrations required to higher values. Since cyclodextrins like MβCD can also extract the SM-sequestered pool of cholesterol at higher concentrations (Das et al., 2014), the most parsimonious interpretation of these data is that PTCH1 reduces the pool of accessible cholesterol in the membrane outer leaflet. Indeed, the effect of PTCH1 mimics the effect of ALOD4 (Figure 2E), a protein known to trap accessible cholesterol (Infante and Radhakrishnan, 2017).

Two different sets of experiments were used to establish that the ability of PTCH1 to antagonize extraction of membrane cholesterol by MβCD was relevant to its biochemical function in Hh signaling (rather than due to an unrelated or off-target effect of PTCH1 overexpression in membranes). First, the inhibitory effect of PTCH1 on MβCD-induced cholesterol extraction could be reversed by the acute administration of SHH in a dose-dependent manner (Figure 3C and G). SHH addition liberated outer leaflet accessible cholesterol in our assay because (1) it restored rapid kinetics of cholesterol extraction by MβCD and (2) its effect could be antagonized by WT ALOD4 (but not a mutant that cannot bind cholesterol) (Figure 3C and H). Second, to establish the specificity of SHH, we turned to PTCH1-ᐃL2, a truncation mutant that cannot bind to Hh ligands (Figure 3A and B). This widely used mutant can act in a dominant fashion to block Hh signaling, even in the presence of Hh ligands (Briscoe et al., 2001). Like WT PTCH1, PTCH1-ᐃL2 also antagonized MβCD-induced cholesterol extraction but, crucially, this effect could not be reversed by SHH (Figure 3I). Interestingly, dose-response analysis demonstrated that PTCH1-ᐃL2 appeared to be more effective than WT PTCH1 in reducing outer leaflet accessible cholesterol (Figure 3—figure supplement 1F). This difference is evident in the absence of Dox, when leaky expression from the Dox-inducible promoter leads to a low amount of PTCH1 expression (Figure 3B). Under these conditions of low-level expression, the t1/2 for cholesterol extraction by MβCD is ~27 s in cells expressing WT PTCH1 (Figure 3C) and ~57 s in cells expressing PTCH1-ᐃL2 (Figure 3I).

If accessible cholesterol is the relevant substrate for PTCH1, expanding the pool of accessible cholesterol should increase the transport burden on PTCH1. Using signaling and differentiation assays, our prior work demonstrated that expanding the accessible cholesterol pool by SM depletion opposes PTCH1 activity: the dose of SHH required to activate signaling is reduced when accessible cholesterol levels are increased (Kinnebrew et al., 2019). In some cell lines, SM depletion even prevented PTCH1 from completely inhibiting SMO in the absence of SHH. To directly test this model, we measured the effect of SM depletion on the ability of PTCH1 to reduce MβCD-induced cholesterol extraction (Figure 4A). As demonstrated previously (Figure 2F), SM depletion increased both the extraction rate and total amount of cholesterol extracted by MβCD. When we expanded the pool of accessible cholesterol, PTCH1 was unable to completely prevent cholesterol extraction by MβCD. Importantly, PTCH1 was still active under these conditions since it reduced both the rate and total amount of cholesterol extracted (Figure 4B). These results show that SM depletion, which markedly potentiates Hh signaling (Kinnebrew et al., 2019), prevents PTCH1 from depleting the membrane outer leaflet of accessible cholesterol and provides an explanation for our previous observation of the opposing effects of PTCH1 and SM depletion on Hh signaling.

Opposing effects of PTCH1 and sphingomyelin (SM) depletion on outer leaflet accessible cholesterol.

(A) Cartoon showing that depletion of SM with myriocin increases accessible cholesterol in the membrane outer leaflet, increasing the transport load on PTCH1 and preventing it from depleting accessible cholesterol from the outer leaflet. (B) Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in the presence (+Dox) or absence (−Dox) of PTCH1 expression and with or without myriocin treatment (80 μM, 3 days) to deplete SM. Each curve shows the mean fluorescence measured from >15 cells taken from at least three biological replicates, with SEM depicted in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data. The t1/2, along with the 95% CI, is shown for each curve. The experiment was repeated three independent times with similar results. CI, confidence interval; SEM, standard error of the mean.

Taken together, these experiments show that PTCH1 depletes accessible cholesterol in the outer leaflet of the plasma membrane in a manner that is regulated by its endogenous ligand SHH.

Requirement of transmembrane ion gradients for the function of PTCH1

PTCH1 has homology to the Resistance Nodulation Division (RND) family of pumps, which use TM proton gradients to efflux toxic molecules out of Gram-negative bacteria (Tseng et al., 1999). While a proton gradient does not exist across the plasma membrane of metazoan cells, PTCH1 is thought to depend on a cation gradient to power its cholesterol transport activity (Ingham et al., 2000; Taipale et al., 2002). However, the identity of the cation gradient used by PTCH1 is disputed, with both the TM sodium (Na+) and potassium (K+) gradients being implicated (Myers et al., 2017; Petrov et al., 2020). Neither of these studies used a direct assay for the biochemical activity of PTCH1; instead, they relied on SMO-induced changes in cAMP (Myers et al., 2017) or SMO accumulation in primary cilia (Petrov et al., 2020) as indirect readouts of PTCH1 activity.

PTCH1 has a triad of acidic residues (D513, D514, and E1095, residue numbers correspond to the human ortholog) that have been implicated in the ability of PTCH1 to inhibit SMO by coordinating the cation that permeates through the center of the TM domain to power its transport activity (Petrov et al., 2020; Taipale et al., 2002; Zhang et al., 2018). Structural studies have noted an extra cryo-EM density consistent with an ion directly coordinated by these residues (Figure 5A; Rudolf et al., 2019). Mutations in these residues impair the ability of PTCH1 to block Hh signaling and have been linked to a familial cancer predisposition syndrome called Gorlin’s syndrome (Taipale et al., 2002). Mutations of these key D513 and D514 residues abrogated the ability of PTCH1 to reduce outer leaflet accessible cholesterol, suggesting that the PTCH1 transport activity we observed in our TIRFM assay may also depend on cation-flux-driven conformational changes (Figure 5B and C). In addition, we were now in a position to address the uncertainty about the identity of the cation gradient required for PTCH1 function using a direct measure of its activity.

PTCH1 cation binding site mutants fail to reduce accessible cholesterol.

(A) A view of the potential ion binding site in the middle of the PTCH1 transmembrane (TM) domain observed in the human SHH-PTCH1 complex (PDB 6DMY4) (Gong et al., 2018). The blue mesh represents the 3.6 Å cryo-EM map (EMD-7968) and shows an extra density, consistent with a potential ion, within binding distance to a cluster of negatively charged residues (D513, D514, and E1095). Two acidic TM residues (present within a GxxxDD motif in TM4 and GxxxE motif in TM10) are conserved in RND family proteins (Petrov et al., 2020; Taipale et al., 2002; Zhang et al., 2018). (B) Western blot showing Doxycycline (Dox)-inducible expression of PTCH1 wild-type (WT) or the D513Y and D513A/D514A mutants. (C) Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in cells expressing the indicated PTCH1 variants (+Dox). The inactivating effect of SHH (1 μM) on WT PTCH1 (red curve) is shown as a control. Each curve shows the mean fluorescence measured from >20 cells taken from at least three biological replicates, with SEM depicted in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data. The t1/2, along with the 95% CI, is shown for each curve. The experiment was repeated three independent times with similar results. CI, confidence interval; SEM, standard error of the mean.

Figure 5—source data 1

Uncropped original scans of films used for immunoblots shown in Figure 5B.

Dotted lines mark the cropped region of the immunoblot that was used to generate panel Figure 5B.

https://cdn.elifesciences.org/articles/70504/elife-70504-fig5-data1-v2.pdf

At rest in metazoan cells, the concentration of Na+ is high on the outside and low on the inside; conversely, the concentration of K+ is high inside and low outside (Figure 6A). The membrane potential (roughly –50 to –70 mV) is closest to the equilibrium potential of K+ since metazoan membranes are most permeable to K+. To test a role for cation gradients in PTCH1 sterol transport, we treated cells with the ionophoric antibiotic monensin, which non-selectively binds monovalent cations, transporting them across the cell membrane in an electroneutral (non-cell-depolarizing) manner. In the presence of monensin, PTCH1 failed to reduce accessible cholesterol in membranes, consistent with a role for monovalent cations in PTCH1 sterol transport (Figure 6B). PTCH1 activity was also blocked by treatment with nigericin, an ionophore that selectively transports K+ ions across the cell membrane and thus dissipates the cellular K+ gradient (Figure 6C).

Figure 6 with 1 supplement see all
PTCH1 activity requires the cellular potassium gradient.

(A) The sodium and potassium gradients present across the plasma membrane of mammalian cells. In each of the panels a red ‘X’ is used to mark the gradient that is disrupted by the treatment used in the panel. Panels (B–F) show time courses of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition (316 μM, arrow) in cells expressing PTCH1 in control media (gray curves) or in media with the indicated additions or substitutions. In each case, SHH has been added (1 μM, red curve) to show the effect of PTCH1 inactivation in control media. (B, C) Effect of monensin (B, 100 μM, 1 hr) or nigericin (C, 80 nM, 30 min). (D, E) Effect of isotonic media in which 150 mM NaCl is replaced with an equal concentration of NMDG-Cl (D) or KCl (E). (F) Effect of hypertonic media containing 150 mM KCl in addition to 150 mM NaCl. Each curve shows the mean fluorescence measured from >20 cells taken from at least three biological replicates, with SEM depicted in lighter shading around each curve. Fluorescence is depicted relative to the starting fluorescence (t/t0) for the TIRFM data. The t1/2, along with the 95% CI, is shown for each curve. The experiment was repeated three independent times with similar results. CI, confidence interval; SEM, standard error of the mean.

To test the role of a Na+ gradient, we incubated cells in a defined, isotonic extracellular medium containing either NaCl (control medium) or medium containing chloride salts of choline+ or N-methyl-D-glucamine (NMDG+). In comparison to Na+, choline+ and NMDG+ are larger cations that are membrane impermeable at the shorter time scales used in our TIRFM assay. Replacement of extracellular Na+ with choline+ or NMDG+ does not alter the K+ gradient and has only a minor effect on the membrane potential (Reuss and Grady, 1979). PTCH1 functioned normally to reduce cholesterol accessibility when extracellular Na+ was replaced with either NMDG+ or choline+, showing that the transport activity of PTCH1 does not depend on Na+ permeation (Figure 6D and Figure 6—figure supplement 1A).

To test a role for the K+ gradient, we used an isotonic extracellular medium in which NaCl was replaced with KCl, abolishing both the TM K+ and Na+ gradients. We also tested the effect of hypertonic medium containing physiological NaCl levels and high extracellular KCl, leaving the Na+ gradient intact but abolishing the K+ gradient. In both cases, the ability of PTCH1 to reduce accessible cholesterol was abolished in the presence of high extracellular K+ (Figure 6E and F). Consistent with a previous study (Petrov et al., 2020), extracellular RbCl and CsCl also inhibited PTCH1 activity in our cholesterol transport assays (Figure 6—figure supplement 1C). Rb+ and Cs+, ions that are larger than K+, may block the ion transport conduit through PTCH1 (Petrov et al., 2020). Taken together, these data show that PTCH1 requires the integrity of the TM K+ gradient to reduce the accessibility of cholesterol in the outer leaflet of the plasma membrane.

Discussion

The discovery that membrane cholesterol is segregated into different pools, made over two decades ago, was partly based on the observation that a fraction of cholesterol in both synthetic and cellular membranes is more susceptible to extraction by MβCD (Haynes et al., 2000; Radhakrishnan and McConnell, 2000). Subsequent characterization has shown that this fraction of membrane cholesterol is likely the same as the fraction that is more accessible to the cholesterol-modifying enzyme cholesterol oxidase and to cholesterol-binding toxins such as PFO and ALO (Das et al., 2014; Gay et al., 2015; Lange and Steck, 2020). These original studies established that the rate of cholesterol extraction by MβCD in both synthetic and cellular membranes serves as a sensitive and quantitative indicator of the size of the accessible cholesterol pool. Building on these previous studies, we established a live-cell, TIRFM assay to measure the impact of expressed proteins (like PTCH1) on accessible cholesterol in the outer leaflet of the plasma membrane. This assay allowed us to directly measure the biochemical activity of PTCH1 in the absence of downstream Hh signaling components like SMO. Our results show that PTCH1 depletes accessible cholesterol in the outer leaflet of the plasma membrane. Conversely, SHH inactivates PTCH1 and elevates outer leaflet cholesterol. More generally, our assay can be readily adapted to study the effect of any protein on membrane accessible cholesterol in the plasma membrane.

The mechanism of cholesterol transport by PTCH1

The numerous PTCH1 cryo-EM structures solved to date give few insights into how PTCH1 may move cholesterol from the outer to the inner leaflet of the plasma membrane. Most of these structural studies have highlighted a hydrophobic tunnel through the extracellular domains of PTCH1 that extends to the outer leaflet of the plasma membrane (Gong et al., 2018; Qi et al., 2018a; Zhang et al., 2020). This tunnel has been proposed to provide a conduit for sterol transport based on the observation of sterol-like densities along its trajectory. Mutations predicted to block this tunnel inhibit PTCH1 activity. When SHH ligands bind to PTCH1, they insert both their attached lipids (the palmitate and the cholesterol) into this tunnel, presumably blocking sterol transport (Qian et al., 2019; Qi et al., 2018b; Rudolf et al., 2019). However, this transport route is inconsistent with two observations: (1) PTCH1-ᐃL2 can suppress SMO activity and Hh signaling in cells and animals (Briscoe et al., 2001) and (2) PTCH1-ᐃL2 can reduce outer leaflet cholesterol accessibility even more effectively than WT PTCH1 (Figure 3—figure supplement 1F). The excision of the entire second extracellular domain PTCH1, which forms one-half of the tunnel, should disrupt transport if the current models are correct. We speculate that the primary function of this PTCH1 tunnel may instead be to accommodate the palmitate and cholesterol moieties attached to SHH, thereby stabilizing an inactive state of the protein, while the TM domain plays a more direct role in inter-leaflet cholesterol flipping. Further structural, functional, and reconstitution studies are required to fully unravel the biochemical mechanism of this important developmental receptor and sterol transporter.

The direction of cholesterol transport by PTCH1

Our observation that PTCH1 reduces accessible cholesterol in the outer leaflet of the plasma membrane raises an important question: what happens to this accessible cholesterol? Three possibilities are as follows (Figure 7A): (1) PTCH1 alters the lateral organization of the membrane and promotes the formation of cholesterol-SM complexes in the outer leaflet, (2) PTCH1 transports this cholesterol out of the cell to an extracellular acceptor, or (3) PTCH1 moves the cholesterol to the inner leaflet or to an intracellular acceptor. Models 1 and 2 predict that PTCH1 should reduce inner leaflet cholesterol (due to the free exchange of cholesterol between the two leaflets), while model three predicts that PTCH1 would increase inner leaflet cholesterol (Figure 7A). We favor model three based on our observation that PTCH1 expression increases the recruitment of two different probes for accessible cholesterol to the membrane inner leaflet (Figure 1). We propose that PTCH1 uses potassium efflux to drive the movement of cholesterol from the outer to the inner leaflet of the plasma membrane, where it is likely then transported to other membrane-bound organelles, including the ER, by sterol transfer proteins. This directionality of transport by PTCH1 is the same as that used by the structurally related lysosomal cholesterol transporter NPC1.

Models for how PTCH1 inhibits SMO by reducing membrane cholesterol.

(A) Three proposed mechanisms by which PTCH1 depletes outer leaflet accessible cholesterol (see text for details). In models 1 and 2, inner leaflet cholesterol is predicted to be reduced, but in model three inner leaflet cholesterol will increase. (B) PTCH1 inhibits SMO by utilizing the cellular potassium gradient to transport outer leaflet accessible cholesterol to the cytoplasmic leaflet (left). When SHH binds to PTCH1, outer leaflet accessible cholesterol rises and activates SMO by binding to its extracellular cysteine-rich domain (right).

Comparison to alternative models for PTCH1 transporter function

Our results diverge from a previous study that proposed that PTCH1 depletes the inner leaflet of cholesterol, thereby preventing cholesterol from entering a tunnel through the center of the SMO transmembrane domain (TMD) (Zhang et al., 2018). Our TIRFM assay is conceptually different from the methods used in this previous study, where steady-state levels of inner leaflet cholesterol were measured by microinjecting cells with a mutant version of the PFOD4 domain that had been modified with environmentally sensitive fluorophores to lower the cholesterol threshold for binding (Liu et al., 2017). These modified PFOD4 probes do not differentiate between accessible and inaccessible pools of cholesterol since their binding to cholesterol-containing vesicles is insensitive to the presence of SM or other phospholipids (Liu et al., 2017). To avoid the complications associated with using these probes to measure membrane cholesterol accessibility (Buwaneka et al., 2021; Courtney et al., 2018; Steck and Lange, 2018), we sought to measure outer leaflet accessible cholesterol using a completely independent method. Our TIRFM assay uses the TdTomato-PFOD4H probe to simply monitor the cholesterol content of the inner leaflet; cholesterol accessibility in the outer leaflet is assessed by the rate of cholesterol transfer from the membrane to MβCD. The propensity of cholesterol to escape from membranes and transfer to a soluble acceptor like MβCD is directly related to its fugacity, which is a classical term for its chemical activity (McConnell and Radhakrishnan, 2003). The chemical activity of cholesterol can be markedly reduced by complex formation with membrane phospholipids like SM. Modulation of cholesterol’s chemical activity by complex formation provides the basis for the accessible and inaccessible pools of cholesterol, which reflect free and complexed cholesterol, respectively. Our kinetic assay, grounded in previous experimental and theoretical work, is a more direct way to measure cholesterol accessibility compared to the steady-state binding of protein probes to membranes, which can be influenced by many extraneous factors (Courtney et al., 2018).

Regulation of Smoothened by membrane cholesterol

Our findings have implications for some of the debates surrounding how SMO is activated in response to SHH. There is uncertainty about whether the cholesterol that binds and activates SMO comes from the inner or outer leaflet of the plasma membrane and about whether cholesterol activates SMO by binding to its extracellular cysteine-rich domain (CRD) or its TMD (Byrne et al., 2016; Deshpande et al., 2019; Huang et al., 2016; Luchetti et al., 2016; Radhakrishnan et al., 2020). Our observation that outer leaflet cholesterol levels rise in response to SHH favors the model that the source of cholesterol that activates SMO is the outer leaflet of the membrane. We propose that cholesterol from the outer leaflet binds to the CRD when PTCH1 is inactivated, driving SMO activation and Hh signaling (Figure 7B). An alternative model for the function of PTCH1 is that it inactivates SMO by directly accepting the cholesterol molecule bound to the SMO CRD (Kong et al., 2019). While our experiments did not test this model directly, our assays in HEK293T cells were performed without SMO co-expression. Thus, PTCH1 is able to change membrane cholesterol organization in the absence of SMO.

The inner and outer leaflets of the plasma membrane are different in their lipid composition (Doktorova et al., 2020; Lorent et al., 2020; Verkleij et al., 1973). Two factors suppress cholesterol accessibility in the outer leaflet: the exclusive presence of SM and the preponderance of phospholipids with saturated acyl chains, which increase interactions with cholesterol (Keller et al., 2000; Lönnfors et al., 2011; Slotte, 1992). In contrast, the inner leaflet has higher levels of acyl chain unsaturation and lower packing, which would be predicted to increase cholesterol accessibility (Lorent et al., 2020). Indeed, our TdTomato-PFOD4H probe was readily recruited to the inner leaflet of the plasma membrane in the absence of any perturbations (Figure 2C). A recent independent study also confirmed that PFOD4-based probes bind to both leaflets of the plasma membrane of HeLa cells (Abe and Kobayashi, 2021). Given these considerations, we propose that the outer leaflet pool of cholesterol, especially in the SM-rich ciliary membrane (Kinnebrew et al., 2019), is a more plausible target of regulation by PTCH1 to control SMO activity. We emphasize that our model for PTCH1 function does not depend on (and does not inform) the outcome of current debates around the distribution of cholesterol between the inner and outer leaflets of the plasma membrane (Buwaneka et al., 2021; Courtney et al., 2018; Liu et al., 2017; Steck and Lange, 2018).

Limitations of this study

A limitation of our assay is that it cannot monitor cholesterol extraction by MβCD selectively in the membrane of primary cilia—the compartment where PTCH1 is thought to inhibit SMO activity (Rohatgi et al., 2007). Consequently, we could not measure the effect of endogenously expressed PTCH1 on membrane cholesterol accessibility, since PTCH1 is largely concentrated in cilia. Instead, we used PTCH1 stably overexpressed throughout the plasma membrane of HEK293T cells, which likely has a different lipid and protein composition compared to the ciliary membrane. However, our previous studies in fixed cells demonstrated that PTCH1 inactivation leads to an increase in labeling of the outer leaflet of primary cilia by fluorescently labeled PFO* (Kinnebrew et al., 2019). Thus, both sets of studies support the model that inactivation of PTCH1 by SHH elevates accessible cholesterol in the outer leaflet of the plasma membrane.

An important question for future research is to understand the cation-driven conformational ch anges that allow cholesterol flipping by PTCH1. While the TM potassium gradient is required for PTCH1 function in cholesterol transport (based on our results) and SMO inhibition (Petrov et al., 2020), these studies did not demonstrate either the direct permeation of potassium through PTCH1 or the requirement of a potassium gradient in a purified assay of PTCH1 activity. Both studies were performed in the complex membrane environment of intact cells, which makes indirect effects impossible to exclude. This is a key issue because the potassium gradient across metazoan membranes is predicted to store less energy than the sodium gradient (since the resting membrane potential is closest to the equilibrium potential of potassium).

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Escherichia coli)BL21 Rosetta DE3 pLYSMilliporeSigmaCat# 71403-3
Cell line (Homo sapiens)293T-REx Flp-InInvitrogenCat# R780-07;RRID:CVCL_U427
Cell line (H. sapiens)293T-REx Flp-In: PTCH1-1D4This paperDr. Rajat Rohatgi (Stanford University)
Cell line (H. sapiens)293T-REx Flp-In: PTCH1-ᐃL2-1D4This paperDr. Rajat Rohatgi (Stanford University)
Cell line (H. sapiens)293T-REx Flp-In: PTCH1-1D4, D513YThis paperDr. Rajat Rohatgi (Stanford University)
Cell line (H. sapiens)293T-REx Flp-In: PTCH1-1D4, D513A/514 AThis paperDr. Rajat Rohatgi (Stanford University)
AntibodyAnti-PTCH1(Rabbit polyclonal)PMID:17641202Dr. Rajat Rohatgi (Stanford University)WB (1:500)
AntibodyAnti-RFP (Mouse monoclonal)Thermo Fisher ScientificCat# MA5-15257; RRID:AB_10999796WB (1:2000)
AntibodyAnti-P38(Rabbit polyclonal)AbcamCat# ab7952; RRID: AB_306166WB (1:2000)
AntibodyAnti-mouse IgG (H + L)(Peroxidase AffiniPure Donkey)Jackson ImmunoResearch LaboratoriesCat# 715-035-150; RRID: AB_2340770WB (1:10,000)
AntibodyAnti-rabbit IgG (H + L) (Peroxidase AffiniPure Donkey)Jackson ImmunoResearch LaboratoriesCat# 111-035-144; RRID: AB_2307391WB (1:10,000)
Recombinant DNA reagentmNeon-ALOD4 in pRSETB (plasmid)PMID:33712199Dr. Arun Radhakrishnan (University of Texas, Southwestern Medical Center)
Recombinant DNA reagentmNeon-ALOD4- mutant in pRSETB (plasmid)PMID:33712199Dr. Arun Radhakrishnan (University of Texas, Southwestern Medical Center)
Recombinant DNA reagentpcDNA5-FRT-TO Flp-In (plasmid)Thermo Fisher ScientificCat# V652020
Recombinant DNA reagentPTCH1-1D4 in pcDNA5-FRT-TO Flp-In (plasmid)This paperDr. Rajat Rohatgi (Stanford University)
Recombinant DNA reagentdL2-PTCH1-1D4 in pcDNA5-FRT-TO Flp-In (plasmid)This paperDr. Rajat Rohatgi (Stanford University)
Recombinant DNA reagentPTCH1-1D4, D513Y in pcDNA5-FRT-TO Flp-In (plasmid)This paperDr. Rajat Rohatgi (Stanford University)
Recombinant DNA reagentPTCH1-1D4, D513A/D514A in pcDNA5-FRT-TO Flp-In (plasmid)This paperDr. Rajat Rohatgi (Stanford University)
Recombinant DNA reagentTdTomato-PFO-D4H in pGEX-6P1 (plasmid)PMID:25663704Dr. Gregory Fairn (University of Toronto)
Recombinant DNA reagentGFP-Gram1b in pEGFP-C1 (plasmid)PMID:31724953Dr. Yasunori Saheki (Nanyang Technological University, Singapore)
Recombinant DNA reagentPOG44 Flp-Recombinase expression vector (plasmid)Thermo Fisher ScientificCat# V600520
Peptide, recombinant proteinSonic HedgehogPMID:19561611Dr. Christian Siebold (Oxford University)
Chemical compound, drugMethyl beta cyclodextrinSigma-AldrichCat# C4555-5G
Chemical compound, drugMyriocinCayman ChemicalsCat# 35891-70-4
Chemical compound, drugDoxycyclineSigma-AldrichCat# D9891
Chemical compound, drugMonensinThermo Fisher ScientificCat# 461450010
Chemical compound, drugNigericinSigma-AldrichCat# 481990
Chemical compound, drugDulbecco’s modified Eagle’s mediumThermo Fisher ScientificCat# SH30081FS
Chemical compound, drugFetal bovine serumSigma-AldrichCat# S11150
Chemical compound, drugSodium pyruvateGibcoCat# 11-360-070
Chemical compound, drugL-glutamineGemini Bio-productsCat# 400106
Chemical compound, drugPenicillin/ streptomycinGemini Bio-productsCat# 400109
Chemical compound, drugNonessential amino acidsGibcoCat# 11140076
Chemical compound, drugEssential amino acidsThermo Fisher ScientificCat# 11130051
Chemical compound, drugVitaminsThermo Fisher ScientificCat# 11120052
Chemical compound, drugGlucoseThermo Fisher ScientificCat# A2494001
Chemical compound, drugHEPESLonzaCat# 17-737E
Chemical compound, drugSodium pyruvateGibcoCat# 11-360-070
Chemical compound, drugHT supplementThermo Fisher ScientificCat# 11067030
Chemical compound, drugOptimemThermo Fisher ScientificCat# 31985-070
Chemical compound, drugPolyethyleniminePolysciencesCat# 23966-1
Chemical compound, drugMatrigelThermo Fisher ScientificCat# CB-40234A
Chemical compound, drugSigmaFast Protease inhibitor cocktail, EDTA-freeSigma-AldrichCat# S8830
Chemical compound, drugHygromycin BVWR Life ScienceCat# 97064-454
Chemical compound, drugNikon Immersion Oil, Type NF 50 cc, nd = 1.515 (23°C)NikonCat# MXA22024
Chemical compound, drugCesium chlorideSigma-AldrichCat# C4036
Chemical compound, drugRubidium chlorideSigma-AldrichCat# R2252
Chemical compound, drugNMDG chlorideSigma-AldrichCat# 66,930
Chemical compound, drugCholine chlorideResearch Products InternationalCat# C41040
OthersChambered #1.5 German Coverglass, 8 wellLab-Tek IICat# 155409

Constructs and plasmids

PTCH1 constructs

Request a detailed protocol

Full-length WT mouse PTCH1, PTCH1-ᐃL2, and the ion-binding site mutants (D513Y and D513A/D514A) were fused to a C-terminal 1D4 tag (amino acid sequence: TETSQVAPA) and cloned into the pcDNA5-FRT-TO Flp-In vector (Thermo Fisher Scientific, Cat# V652020) to enable inducible expression in the 293T-REx Flp-In cell system. PTCH1-ᐃL2, which carries a deletion of the second extracellular loop (L2, amino acids 793–994), was a gift from James Briscoe (Briscoe et al., 2001).

ALOD4 constructs

Request a detailed protocol

Plasmids encoding His6-mNeon-FLAG-ALOD4 (designated as ALOD4, mNeon-ALOD4, or WT ALOD4) and His6-mNeon-FLAG-ALOD4-mutant (designated as ALOD4-mutant) have been previously described (Johnson and Radhakrishnan, 2021). The ALOD4 used in these constructs (amino acids 404-512 of Anthrolysin O) contains two point mutations (S404C and C472A) that do not affect its activity. The cholesterol-binding mutant of ALOD4 contains six additional mutations (G501A, T502A, T503A, L504A, Y505A, and P506A) that abrogate cholesterol binding (Endapally et al., 2019). All constructs were cloned into the pRSETB vector for bacterial expression.

Purification of SHH ligands

Request a detailed protocol

Human SHH carrying two isoleucine residues at the N-terminus and a hexahistidine tag at the C-terminus (known as SHH-C24II) was expressed in Escherichia coli Rosetta(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration chromatography as described previously (Bishop et al., 2009). The two isoleucines at the N-terminus of SHH-C24II mimic the palmitate attached to native SHH. SHH-C24II, which otherwise lacks both the palmitate and cholesterol modification found in the native ligand, has been validated as an easy to purify and well-behaved substitute for the endogenous ligand (Taylor et al., 2001).

mNeon-ALOD4 protein expression and purification

Expression

Request a detailed protocol

ALOD4 plasmids were transformed into E. coli competent cells (BL21 [DE3] pLysS, MilliporeSigma, Cat #71403) and plated on Luria Broth (LB) agarose plates containing ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml). A single colony was picked and grown overnight in 160 ml LB containing ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml) and used to inoculate a 1 L culture of LB. When the culture reached an OD600 of 0.4–0.6, it was cooled to 18°C and protein expression was induced with 1 mM IPTG at 18°C for 18–24 hr. Cells were harvested at 3500×g for 10 min at 4°C, and immediately used for purification.

Purification

Request a detailed protocol

All steps of the purification were performed at 4°C or on ice. Cell pellets were resuspended in 20 ml of ice-cold Buffer A (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM TCEP) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitor (SigmaFast Protease inhibitor cocktail, EDTA-free; Sigma-Aldrich, Cat#S8830), and 1 mg/ml lysozyme and lysed by 10–15 passes through an EmulsiFlex C5 (Avestin) homogenizer. The Lysate was then clarified by centrifugation (30 min, 25,000×g) and incubated on an orbital shaker with 1 ml of Ni-NTA resin for 30 min. The protein-resin mixture was poured into a chromatography column to collect the flow through. The 1 ml packed column was washed with 50 ml of Buffer A and then 50 ml of Buffer A supplemented with 25 mM imidazole. Bound protein was eluted with Buffer A containing 250 mM imidazole in 5×1 ml fractions. Peak eluate fractions were pooled, concentrated (Amicon Ultra-4 10 kDa cutoff centrifugal filter), and loaded onto a Superdex 200 gel filtration column (Amersham Biosciences) equilibrated with Buffer A. Fractions containing pure His6-mNeon-Flag-ALOD4 (as judged by coomassie staining) were pooled, concentrated, aliquoted, and then stored in 20% glycerol at –80°C. Immediately before use in experiments, an aliquot was thawed on ice, diluted to 5 µM in 0.5% serum Dulbecco’s modified Eagle’s medium (DMEM), and then added to live cells. See sections on Cell Culture and Drug Treatments for further details.

Mammalian cell lines

Request a detailed protocol

293T-REx Flp-In cells were purchased from Invitrogen (Cat# R780-07). These purchased cell lines came with a certificate of authentication from the vendor and were used without further validation. All stable cell lines (see below) derived from the 293T-REx Flp-In cells were validated by Western blotting for the stably expressed protein. Cell lines were confirmed to be negative for Mycoplasma infection.

Cells were grown in high glucose DMEM (Thermo Fisher Scientific, Cat# SH30081FS) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, Cat# S11150) and the following supplements (hereafter called supplemented DMEM): 1 mM sodium pyruvate (Gibco, Cat# 11-360-070), 2 mM L-glutamine (Gemini Bio-products, Cat# 400106), 1× MEM nonessential amino acid solution (Gibco, Cat# 11140076), penicillin (40 U/ml), and streptomycin (40 µg/ml) (Gemini Bio-products, Cat# 400109). Supplemented DMEM was sterilized through a 0.2 μm filter and stored at 4°C. For TIRFM and Western blotting experiments, cells were seeded in 10% FBS supplemented DMEM and then grown to 90–100% confluency. Cells were serum starved in 0.5% FBS supplemented DMEM for 16 hr prior to the start of experiments.

Stable cell line generation

Request a detailed protocol

293T-REx Flp-In cells (Invitrogen, Cat# R780-07) expressing PTCH1 were generated using PTCH1 constructs cloned into the pcDNA5 FRT-TO Flp-In vector. 293T-REx Flp-In cells were seeded in 10 cm cell culture dishes in 10% FBS supplemented DMEM lacking penicillin and streptomycin, grown to 75% confluency and transfected with a mixture of 750 ng of the pcDNA5 FRT-TO construct and 5.4 μg of the POG44 Flp-Recombinase expression vector (Thermo Fisher Scientific, Cat# V600520) using polyethyleneimine (PEI, linear transfection grade; Polysciences Cat# 23966-1). A ratio of 4 μl PEI: 1 μg of total DNA was prepared in 400 μl of room temperature OptiMEM (Thermo Fisher Scientific, Cat# 31985-070), mixed briefly by vortexing, and then incubated at room temperature for 15 min before adding dropwise to cells. After 24 hr, media was aspirated and replaced with 10% FBS supplemented media containing penicillin (40 U/ml) and streptomycin (40 μg/ml). After 24 hr, media was exchanged to fresh 10% FBS supplemented DMEM containing 2 μg/ml Hygromycin B (VWR Life Science, Cat# 97064-454). The majority of cells (>95%) died with Hygromycin B selection, and media was exchanged periodically to remove dead cells.

To confirm expression of PTCH1 variants, each respective cell line was seeded in 2× 6 cm dishes in 10% FBS supplemented DMEM. Cells were grown to 90–100% confluency and then media was exchanged to 0.5% FBS supplemented DMEM. In one 6 cm dish, PTCH1 expression was induced by adding 1 µM Doxycycline. After 16–24 hr, cells were scraped off the plate in 4°C 1× phosphate-buffered saline and collected by centrifugation at 1000×g. Cells were lysed in 150 mM NaCl, 50 mM Tris-HCl pH 8, 10% NP-40, 1× protease inhibitor (SigmaFast Protease inhibitor cocktail, EDTA-free; Sigma-Aldrich, Cat#S8830), 1 mM MgCl2, and 10% glycerol. Lysate was clarified by spinning at 20,000×g for 30 min and 4°C and then protein concentration was measured with a BCA assay. Equal amounts of protein were taken for each sample and incubated with 1 mM TCEP and 1× Laemmli buffer for 30 min at 37°C. Samples were then subjected to SDS/PAGE and finally blotted for expression of PTCH1 using a PTCH1 antibody raised against the PTCH1 cytoplasmic tail (Rohatgi et al., 2007) and P38 (anti-P38 rabbit polyclonal; Abcam, Cat# ab7952; RRID: AB_306166) as a loading control.

Live cell imaging with total internal reflection fluorescence microscopy

Cell preparation

Request a detailed protocol

All TIRFM experiments were carried out in live-cell imaging chambers (Lab-Tek II Chambered #1.5 German Coverglass, eight-well, Cat# 155409) on a Nikon TIRF system. To aid in cell adherence, the live cell imaging chambers were first prepared by coating with Matrigel (Corning 356234). Matrigel was diluted 1:20 in ice-cold 0.5% FBS supplemented DMEM and 100 μl was added to coat the bottom of each well. After allowing Matrigel to solidify for 40 min at room temperature, chambers were washed once with 37°C 10% FBS supplemented DMEM prior to cell plating.

Cells were counted and plated in 300 μl per well at a density of 90,000 cells per well. After 24 hr, or at 90% confluency, cells were transfected with TdTomato-PFOD4H. A transfection reaction for one well included 100 ng TdTomato-PFOD4H and 0.4 μl PEI diluted (ratio of 4 μl PEI: 1 μg of total DNA) into 15 μl of OptiMEM (Thermo Fisher Scientific, Cat# 31985-070) (typically at least eight wells were transfected simultaneously, and transfection reactions were prepared in one 8× mixture). Transfection reactions were briefly vortexed and then incubated at room temperature for 15 min. The transfection reaction was then diluted into 37°C 0.5% FBS supplemented DMEM at a sufficient volume to add 300 μl to each well of the live-cell chamber. Finally, the media from each well-containing cells was aspirated, and 300 μl of transfection mixture was added. If PTCH1 expression was desired, doxycycline was added to a final concentration of 1 μM directly to the diluted transfection mixture. Approximately 16 hr later, live-cell imaging was performed. Immediately before imaging, culture media was replaced with 300 μl 37°C 0.5% FBS supplemented DMEM.

Microscopy

Request a detailed protocol

The TIRF microscope live-cell imaging chamber was warmed to 37°C prior to imaging. Cells were imaged with a Nikon Apo TIRF 100×/1.49 Oil objective (W.D. 0.12, coverglass thickness 0.13–0.2). TdTomato-PFOD4H fluorescence was excited with a 561 nm laser and GFP-GRAM1b fluorescence was excited with a 491 nm laser. All imaging was carried out using MicroManager software (https://micro-manager.org/). First, oil was added to the objective (Nikon Immersion Oil, Type NF 50 cc, nd = 1.515 (23°C); Cat# MXA22024) and then cells were located. An optimum TIRF angle was established. Multidimensional acquisition parameters were set such that an image was collected every 2 s, for 150 frames (300 s total). Once a movie was initiated, SHH was added at 1 μM at 20 s (frame 10) when indicated, and MβCD was added at 316 μM (unless otherwise stated) at 100 s (frame 50).

Steady-state measurements of TdTomato-PFOD4H and GFP-GRAM1b were captured by first locating cells and identifying an optimum TIRF angle. Fields of cells were selected if they had at least five cells. Fluorescence was captured using the MicroManager software ‘Snap Shot’ function, and then each image was saved for downstream analysis (see next section).

TIRFM analysis

Request a detailed protocol

TIRFM movies were analyzed in Fiji2 using a Time Series Analyzer plugin (https://imagej.nih.gov/ij/plugins/time-series.html). To quantify changes in TdTomato-PFOD4H fluorescence, a region of cell membrane was selected that made up ~20–50% of the total surface area of a cell. Each field of cells contained more than five cells, enabling >5 measurements per movie. At least three movies were generated per treatment condition and each experiment was repeated at least three independent times on separate days. Once an area to be measured was chosen, it was added to the Time Series Analyzer window. The average fluorescence of each selected area was then calculated using the ‘Get Average’ function, returning a matrix containing the average fluorescence values for each selected region at each time point for a given movie. The average fluorescence values for each chosen area were then normalized to the starting average fluorescence (t/t0), such that the starting fluorescence value equaled one. Finally, values were plotted in GraphPad Prism 9.1 with time in seconds on the x-axis and t/t0 average fluorescence values on the y-axis. Raw curves, without normalization to the baseline, are shown for selected experiments in Figure 2—figure supplement 1D and Figure 3—figure supplement 2. Each circle making up a point on a curve represents the mean TdTomato-PFOD4H fluorescence at that time point, and the standard error of the mean is depicted as the shading around the curve. The number of cells measured for each experiment is stated throughout the text in the figure legends. Curve fitting was performed in GraphPad Prism 9.1 using the nonlinear regression curve fit for a ‘plateau followed by a one phase exponential decay.’ The time taken to reach half-maximal fluorescence (t1/2) for each curve is reported in the figures, with the upper and lower 95% confidence interval bounds denoted in parentheses.

Analysis of steady-state TdTomato-PFOD4H and GFP-GRAM1b fluorescence was carried out in Fiji2. A region of cell membrane was selected that made up ~20–50% of the total surface area of the cell. The average signal intensity for that region was measured with the ‘Ctrl-m’ function of Fiji2. Values were then plotted without normalization in GraphPad Prism 9.1 using a column graph showing individual values. Outliers were excluded using the Identify Outlier function of GraphPad Prism 9.1 (ROUT method with a Q-score=10%).

Drug treatments for total internal reflection fluorescence microscopy

MβCD treatment

Request a detailed protocol

Methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich, Cat# C4555-5G) was diluted immediately prior to experiments in filtered milliQ water to generate a 38 mM stock. During imaging, MβCD is added directly to cells at indicated concentration without prior dilution.

ALOD4 treatment

Request a detailed protocol

To deplete accessible cholesterol with ALOD4, cells were seeded in 10% FBS supplemented DMEM and grown to 90–100% confluency. Cells were then transfected with TdTomato-PFOD4H in 0.5% supplemented DMEM (see section on Live-cell imaging with TIRFM) for 16 hr. Finally, low serum supplemented DMEM was exchanged for 0.5% supplemented DMEM containing 5 μM mNeon-ALOD4 or 5 μM of the mNeon-ALOD4 cholesterol-binding mutant. Cells were then returned to the cell culture incubator (37°C and 5% CO2) for 45 min. Immediately before imaging, media containing ALOD4 was removed and replaced with 37°C 0.5% supplemented DMEM.

Sphingomyelin depletion with myriocin

Request a detailed protocol

Cells were seeded in 10% FBS supplemented DMEM at an initial density that allowed for 3 days of growth prior to experimentation. After the cells adhered to Matrigel-coated live cell imaging chambers (1–2 hr), media was removed and replaced with 10% FBS supplemented DMEM containing 80 μM myriocin. 16–24 hr before imaging, cells were transfected with TdTomato-PFOD4H (see section on Live-cell imaging with TIRFM) in 0.5% FBS supplemented DMEM containing fresh 80 μM myriocin.

Nigericin and monensin treatments

Request a detailed protocol

Prior to live-cell imaging, media were removed from each well of the live-cell imaging chambers and replaced with 37°C 0.5% FBS supplemented DMEM containing 80 nM nigericin (Sigma-Aldrich, Cat# 481990) for 30 min or 100 μM monensin (Thermo Fisher Scientific, Cat# 461450010) for 1 hr.

Ion gradient treatments

Request a detailed protocol

To test the effect of various ions, Base Media was prepared with the following components: 1× essential amino acids (Thermo Fisher Scientific, Cat# 11130051), 1× MEM nonessential amino acid solution (Gibco, Cat# 11140076), 2 mM L-glutamine (Gemini Bio-products, Cat# 400106), 1× vitamins (Thermo Fisher Scientific, Cat# 11120052), 1× glucose (Thermo Fisher Scientific, Cat# A2494001), 50 mM HEPES (Lonza, Cat# 17-737E), 1 mM sodium pyruvate (Gibco, Cat# 11-360-070), 1× HT supplement (Thermo Fisher Scientific, Cat# 11067030), 2 mM CaCl2, 1 mM MgSO4, and 0.5% FBS. Base Media was sterilized through a 0.2 μm filter and stored at 4°C. Immediately prior to experiments, media was supplemented with salts at specified concentrations denoted in the figure legends. For isotonic control media, NaCl was added to a final concentration of 150 mM and KCl was added to 5 mM.

Statistical analysis

Request a detailed protocol

Data analysis and visualization were performed in GraphPad Prism 9.1. Model figures (Figures 1A4A6A, 7A and B) were made in Adobe Illustrator CS6. TIRFM and epifluorescence images (Figure 2C) were rendered in Fiji2 and the Patched one structure (Figure 5A, PDB 6DMY4) was generated in PyMOL. Scatter dot plots (Figure 1B–C, Figure 1—figure supplement 1A and Figure 2—figure supplement 1A, B) were generated with default settings in GraphPad Prism 9.1; outliers were excluded using the Identify Outlier function of GraphPad Prism 9.1 (ROUT method with a Q-score=10%). Median and interquartile ranges for each plot are denoted by horizontal and vertical lines, respectively.

All statistical analyses used nonparametric methods, which do not assume an underlying normal distribution in the data. The statistical significance of differences between two groups (Figure 1B–C, Figure 1—figure supplement 1A and Figure 2—figure supplement 1A, B) was determined by the Mann-Whitney test. Information about error bars, statistical tests, p-values and n values are reported in each figure legend and were calculated using GraphPad Prism 9.1. All experiments included at least three independent trials with consistent results, unless otherwise noted in the figure legend.

Throughout the paper, the numerical p-values for the comparisons from GraphPad Prism 9.1 are given in the figure legends and denoted on the graphs according to the following key: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, non-significant (ns) p>0.05.

Data availability

No dataset was generated or used during this study (such as deep sequencing data, mass spectrometry data, structural coordinates or maps, genetic data or clinical trial data) that required deposition in a repository such as GenBank, the PDB, mass spec data repositories, or clinical data repositories. We have provided original, uncropped scans of immunoblots shown in Figures 2B, 4B, and Figure 3-figure supplement 1 in the Source Data Files. All other data generated are included in this study, with replicates and statistics described in the figure legends and methods.

References

    1. Christian AE
    2. Haynes MP
    3. Phillips MC
    4. Rothblat GH
    (1997)
    Use of cyclodextrins for manipulating cellular cholesterol content
    Journal of Lipid Research 38:2264–2272.
    1. Tseng TT
    2. Gratwick KS
    3. Kollman J
    4. Park D
    5. Nies DH
    6. Goffeau A
    7. Saier MH
    (1999)
    The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins
    Journal of Molecular Microbiology and Biotechnology 1:107–125.

Decision letter

  1. Duojia Pan
    Reviewing Editor; UT Southwestern Medical Center and HHMI, United States
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States

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

Decision letter after peer review:

Thank you for sending your article entitled "Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes" for peer review at eLife. Your article is being evaluated by 2 peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor.

This paper addresses an important question regarding the mechanisms of Hedgehog signaling. The authors develop a new method to observe changes in cholesterol accessibility in the outer lamella of the plasma membrane to investigate the activity of the Hedgehog receptor PTCH1 and its modulation by Sonic Hedgehog. The results support the conclusion that PTCH1 needs a potassium gradient to reduce chemically-active cholesterol in the outer lamella, presumably by translocating the sterol to the inner lamella. The proposed model contradicts previous reports that suggest transport in the opposite direction using the plasma membrane sodium gradient for energy. While the reviewers appreciate the potential impact of the findings, a major limitation of this study is the reliance on overexpressed PTCH1. The reviewers and editors feel that demonstrating a similar activity for endogenous PTCH1, for example by comparing endogenous PTCH1 with or without SHH ligand or Ptch1+/+ vs Ptch1-/- cells, is essential.

Reviewer #1 (Recommendations for the authors):

This manuscript investigates how the Hedgehog receptor protein Patched 1 (PTCH1) controls signaling activity of the transducer protein Smoothened (SMO). Studies follow up on a previous eLife publication from Rohatgi and Seibold labs suggesting PTCH1 represses SMO by limiting accessible cholesterol in cellular membranes. Cholesterol is proposed to function as the SMO agonist through binding a site in its extracellular cysteine rich domain (CRD), and that PTCH1 governs SMO activation by preventing accessible membrane cholesterol from entering this site.

A pro of the study is the development and validation of a quantitative fluorescent assay to monitor available membrane cholesterol. This assay measures accessible cholesterol through TIRF imaging of a fluorescent cholesterol binding protein (PFO) that associates with accessible (non-sphingomyelin associated) membrane cholesterol. In each experiment, MβCD is used to extract cholesterol from the outer membrane, which leads to flipping of cholesterol from inner to outer plasma membrane. Depletion of cholesterol from the inner membrane releases PFO to the cytoplasm, which decreases TIRF signal. This assay is the first to measure 'enzymatic' activity by PTCH1 in a quantifiable way. In prior studies, PTCH1 control of membrane cholesterol was inferred by tracking SMO ciliary localization, which is inhibited by active PTCH1. In addition to this SHH-centric benefit, the assay will likely be useful for other research applications necessitating quantification of available membrane cholesterol.

Using the PFO TIRF assay, the authors provide evidence that PTCH1 reduces accessible cholesterol and that the ligand SHH blocks this activity. Use of additional cholesterol binding probes allowed the authors to propose a model in which PTCH1 flips cholesterol from the outer to inner plasma membrane. They speculate that inner leaflet cholesterol is then transported by an intracellular cholesterol binding protein to shuttle it away from the membrane, thus preventing its association with SMO.

Despite the methodological advance of the PFO TIRF assay, the study only modestly advances knowledge beyond what has already been reported by this group and others – that PTCH1 moves cholesterol (Rohatgi, Seibold, Beachy, Salic Labs) and that it requires the K+ gradient to do so (Salic Lab). However, since the manuscript is being considered as an eLife Research Advance, rather than a Research Article, I am supportive of publication.

I don't disagree with the data interpretations but am having a hard time visualizing the biology of the system when PTCH1 is expressed because of the way the MβCD results are presented. The gist is that there is less free cholesterol in the outer membrane when PTCH1 is around, so MβCD doesn't have much to extract and PFO TIRF is not redistributing. However, since the lines don't shift following MβCD (Figure 2C as an example), it looks like cholesterol is locked in the membrane by PTCH1, rather than already having been depleted. I think this is a visualization problem because everything is normalized to 1. Are the baseline readings significantly different between the samples? Is baseline higher/lower when PTCH1 is expressed? I needed to sketch it out for myself, and I feel like this is too much work for the reader. Is there a way to convey the results so they're more intuitive? More cartoons, maybe? The cartoons provided are very helpful.

Re: Figure 6 and related to the point above: If PTCH1 is flipping cholesterol to the inner membrane, this should be reflected by PFO TIRF even in the absence of MβCD. Do you see a higher baseline in response to Dox induction of PTCH1 expression? The results presented in Figure 6 are less convincing that the rest of the figures, so it felt like the manuscript was ending with an afterthought. This point would be improved by additional experimental support and more discussion. There is only one sentence discussing the results in Figures 6B and C.

Reviewer #2 (Recommendations for the authors):

Recent publications suggest that PTCH1 is a cholesterol transporter that mobilises cholesterol or a cholesterol derivative from the inner leaflet of the plasma membrane to the outer leaflet, where its concentration is about 10-fold higher (Zhang et al., 2018, Cell 175, 1352-1364). This transport against a concentration gradient requires energy, and previous literature indicates that sodium is the cation that provides such energy (Myers et al., Proc Natl Acad Sci U S A 2017;114(52):E11141-E11150). PTCH1 contains an acidic triad (EED), conserved in bacterial RND permeases, that is essential for its activity as Smoothened repressor and likely allows co-transport of the cation.

In this study, Kinnebrew et al., follow their previous finding that indicates that sphingomyelin (SM) depletion potentiates Hedgehog signalling (Kinnebrew et al., eLife. 2019 Oct 30;8:e50051). SM localises in the outer leaflet of the plasma membrane and forms a complex with cholesterol, reducing availability of "free" or chemically-active cholesterol. Given that this suggests that increases in cholesterol concentration in the outer leaflet drives Smoothened activation, as opposed to the findings of the Beachy Lab, they re-evaluate PTCH1's transport directionality in the current study using total internal reflection fluorescence microscopy (TIRFM) to image reduction of binding of a cholesterol sensor domain fused to a fluorescent protein (TdTomato) to the inner leaflet when cholesterol is extracted from the outer lamella using methyl-β-cyclodextrine. The findings support the author's conclusion that PTCH1 reduces accessible outer leaflet cholesterol, but several methodological and conceptual questions remain:

– MβCD pinches accessible cholesterol out of the membrane, but it is unclear if the rate of extraction is comparable to the unknown rate of transport by PTCH1, or if a rate-limiting aspect of the model confounds the interpretation of the rate of PFOD4H-TdTomato fluorescence reduction. Therefore, while the assay can be optimal to capture spontaneous flip-flopping, it may not be a faithful readout of a facilitated transport event.

– PFOD4H-TdTomato fluorescence in the steady-state is more likely to represent the inner leaflet cholesterol content. Because the TIRFM signals are normalised to t=0, this crucial information is not available in most experiments other than in Figure 6. This would be particularly useful in cells expressing PTCH1 and PTCH1-DL2, and after a few minutes of SHH addition, as the affinity of PFOD4H for cholesterol is close to the reported inner leaflet cholesterol level (~ 1-3 mol%).

– If myriocin treatment increases accessible cholesterol in the outer leaflet, wouldn't it increase the inner leaflet cholesterol content by flip-flop? If that is the case, the non-normalised TIRF signal should be higher at t=0 than in vehicle-treated cells.

– The concentration of SHH ligand used to module cholesterol availability seems excessively high (10-6 M). The same group used 25 nM in the previous study as a "high, saturating SHH concentration", in line with most groups using SHH in the range of 50-100 nM. A dose-response effect will also inform if the IC50 of SHH in the TIRF assay agrees with its Kd for PTCH1 and the EC50 for stimulation of Gli-dependent transcription.

– The lipid modifications of SHH play a key role in the asymmetric binding mode to PTCH1 dimers. The article does not detail the modifications of the SHH used.

– Induction of PTCH1 expression increases absolute TIRF signals in Figure 6, suggesting higher inner leaflet cholesterol. However, this interpretation depends on equal total fluorescence, i.e. equal expression of the fluorescent sensor proteins regardless of PTCH1 expression and SHH signalling.

– If PTCH1 reduces accessible cholesterol in the outer lamella, one would expect to observe a reduction in binding of an extracellular cholesterol sensor, equivalent to the PFOD4H-TdTomato.

– The biggest limitation of this study is the reliance on overexpressed PTCH1. The effect of SHH addition to Hh-competent cells expressing endogenous PTCH1 (such as NIH 3T3 cells) on cholesterol sensor measurements is essential, even if it cannot be determined in the primary cilium membrane.

– Conceptual concern: why would PTCH1 require energy provided by a cation gradient to move cholesterol in favour of its concentration gradient? The related cholesterol transporter NPC1 transports cholesterol from the outer to inner leaflet without an energy gradient, and it lacks the acidic triad essential for PTCH1 activity.

– This study clashes with previous reports using confocal microscopy vs TIRF. One question that comes to mind is if a different conclusion would be drawn using the PFOD4H-TdTOmato sensor in standard confocal imaging to image changes in lateral membranes that are more readily exposed to solvent and SHH than the basal membrane attached to the substratum.

– The contradictory findings are unlikely to be explained simply by a difference in the cholesterol sensor used (PFOD4H-TdTOmato vs PFOD4H tagged with a small solvatochromic fluorophore). Additional controls and a deeper discussion of the geometry of the assay and the potential impact of measuring MβCD-induces changes in fluorescence vs steady-state levels will be necessary to understand the differences.

However, I identified some areas that need to be addressed. I would like you to consider providing additional evidence for some of my key concerns:

1 – Provide baseline absolute levels of fluorescence in each condition.

2 – Demonstrate that induction of PTCH1 does not affect total expression of PFOD4H-TdTomato.

3 – Investigate the effect of adding more physiological concentrations of Shh (in the 10-50 nM range) to Hh-competent cells expressing the cholesterol sensor. Potential controls of acute silencing of PTCH1.

4 – Testing changes in binding of an extracellular cholesterol sensor in the system.

5 – Indicate source and molecular details of the SHH ligand used: is it lipidated, is the Ile-Ile mimic of the N-terminal palmitate? Please add this to the methods.

If Shh is unmodified, could you test if dual lipidated SHH has the same effects at a lower concentration?

6 – I'd also like to see a deeper discussion on the potential explanations of the Zhang et al., results. You are creating a lively controversy in the field and would strongly benefit from trying to figure out why your conclusions are correct and the other is not when the systems are so similar in most ways.

https://doi.org/10.7554/eLife.70504.sa1

Author response

This paper addresses an important question regarding the mechanisms of Hedgehog signaling. The authors develop a new method to observe changes in cholesterol accessibility in the outer lamella of the plasma membrane to investigate the activity of the Hedgehog receptor PTCH1 and its modulation by Sonic Hedgehog. The results support the conclusion that PTCH1 needs a potassium gradient to reduce chemically-active cholesterol in the outer lamella, presumably by translocating the sterol to the inner lamella. The proposed model contradicts previous reports that suggest transport in the opposite direction using the plasma membrane sodium gradient for energy. While the reviewers appreciate the potential impact of the findings, a major limitation of this study is the reliance on overexpressed PTCH1. The reviewers and editors feel that demonstrating a similar activity for endogenous PTCH1, for example by comparing endogenous PTCH1 with or without SHH ligand or Ptch1+/+ vs Ptch1-/- cells, is essential.

This important point, which is the basis of our current manuscript, has been demonstrated in Figure 8 of the parental paper for this Research Advance (Kinnebrew et al., 2019). A key fact relevant to this question is that the bulk of endogenous PTCH1 in NIH/3T3 cells is localized in the ciliary membrane, a miniscule subcompartment of the plasma membrane (Rohatgi et al., 2007). In our prior study, we used the steady-state binding of fluorescently-labeled PFO* to measure outer leaflet accessible cholesterol either over the whole plasma membrane (by FACS, Figure 8A) or selectively at primary cilia (by microscopy, Figure 8B) in NIH/3T3 cells expressing endogenous PTCH1. SHH induced an increase in outer leaflet PFO* binding at the ciliary membrane (Figure 8B), consistent with the data from TIRFM assays presented in the current manuscript. Importantly, SHH did not change PFO* binding to the overall plasma membrane (Figure 8A) in NIH/3T3 cells, since PTCH1 localizes and functions at primary cilia (not all over the plasma membrane). Our TIRFM assay reports on the entire plasma membrane, not specifically on the ciliary membrane. Consequently, our TIRFM assay does not detect any changes in outer leaflet cholesterol accessibility in NIH/3T3 cell or Mouse Embryonic Fibroblasts (MEFs) after treatment with SHH (a result completely consistent with our 2019 elife paper showing that PFO* binding to the outer leaflet of the plasma membrane in NIH/3T3 cells is unchanged by SHH). In conclusion, the results of steady-state PFO* binding in NIH/3T3 cells shown in our 2019 elife paper are in agreement with the TIRFM assay used in the current manuscript. In the context of both endogenous PTCH1 (2019 paper) and overexpressed PTCH1 (current manuscript), SHH causes an increase in outer leaflet cholesterol accessibility.

We agree that using endogenously expressed proteins is preferable whenever possible. However, we respectfully take the position that model assays using overexpression in heterologous systems have played an irreplaceable role in elucidating the molecular mechanism of many transporter proteins and channels (whose biochemical activities would have been otherwise very difficult to directly characterize due to low or highly compartmentalized expression). A prominent example is the extensive use of mRNA injection in Xenopus laevis oocytes to overexpress and then characterize functions of diverse mammalian and even plant membrane proteins (Pike et al., 2019; Wagner et al., 2000). Other examples include the use of overexpression in HEK293T and Cos cells to study the mechanism of other cholesterol/sterol transporters, including ABC1 and the scavenger receptor SR-BI (Acton et al., 1996; Wang et al., 2001). Perhaps the most relevant example in the context of our manuscript are studies that demonstrated that PTCH1 is the direct receptor for SHH. Biochemical evidence that SHH binds to PTCH1 required the use of overexpression: in Xenopus laevis oocytes by Cliff Tabin’s lab (Marigo et al., 1996) or HEK293 cells by Arnon Rosenthal’s lab (Stone et al., 1996). Due to low endogenous PTCH1 expression levels, it is impossible to detect differences in SHH binding to wildtype and Ptch1-/- MEFs.

Of course, overexpression studies have to be well-controlled and we provide several types of controls to ensure that the effects we see are physiologically relevant. Interfering with PTCH1 biochemical function using three completely different (but well-established) strategies-- a classical point mutation (D513Y, Figure 5), addition of its known inactivating ligand SHH (Figure 3) or dissipation of the K+ gradient (Figure 6)-- abolishes its effect on outer leaflet cholesterol. These controls exclude the possibility that the effects we observe are a non-specific artefact of overexpression.

In response to this comment, we have added a section to the Discussion titled “Limitations of this study” (lines 385-402) that thoroughly discusses the limitations of this assay, including the use of PTCH1 overexpression and the inability to selectively measure extraction of ciliary cholesterol. In addition, we conducted two experiments whose results are shown below:

To understand the degree of PTCH1 overexpression in our system, we used immunofluorescence to measure the abundance of PTCH1 at the plasma membrane of our HEK293T cells after Dox addition and compared it to the abundance of PTCH1 at primary cilia in a Hh-responsive, ciliated mouse embryonic fibroblast (MEF) cell line we have extensively used in prior publications to study the function of PTCH1 (Rohatgi et al., 2007). As shown in Author response image 1, the abundance of PTCH1 at primary cilia is comparable to the abundance of PTCH1 at the plasma membrane in the HEK293T cells used in our manuscript. Thus, the abundance of PTCH1 in the HEK293T cells used in our current manuscript is comparable to other systems where PTCH1 function, localization and signaling have been studied.

Author response image 1

Anti-PTCH1 antibodies were used to measure the fluorescence of PTCH1 at primary cilia in mouse embryonic fibroblasts (MEFs) stably expressing PTCH1 (left) or at the plasma membrane of HEK293T cells used in our manuscript after Dox treatment (16 hours, 1 micromolar). Each dot represents one cilium (MEFs) or one cell (HEK293T cells). To show that the anti-PTCH1 IF signal is specific, PTCH1 measurements were also performed in MEFs treated with SHH (100 nM) for 16 hours (which is known to clear PTCH1 from cilia) and in HEK293T cells without Dox exposure.

As we suggested in the revision plan, we exposed cells to lower concentrations of Dox to see if PTCH1 could be induced to lower levels (Author response image 2) . As shown by the immunoblot (top), the dynamic range is limited, PTCH1 abundance rapidly increases between 0.1 nM and 10 nM of Dox. We conducted TIRFM assays to measure outer leaflet cholesterol accessibility at various concentrations of Dox (0.1-1 nM), with the results shown in (Author response image 2) (bottom). As we increase the Dox concentration from 0.1 to 0.3 to 0.9 nM, we observed a progressive decrease in outer leaflet accessible cholesterol-- shown by both a decrease in the initial extraction rate and a decrease in the maximum amount of cholesterol extracted. Thus, within the limitations of our Dox-inducible system, the influence of PTCH1 on outer leaflet cholesterol accessibility is dose-dependent and the effects are observable at significantly lower abundances of PTCH1. Additionally, new data in the revised manuscript (Figure 3G) shows that the effect of SHH (an inactivating ligand for PTCH1) on outer leaflet cholesterol is dose-dependent.

Author response image 2
Time course of the change in TdTomato-PFOD4H membrane fluorescence after MβCD addition to HEK293T cells treated with increasing concentrations of Dox to induce the expression of progressively greater amounts of PTCH1.

The immunoblot (top) shows whole cell PTCH1 abundance after treatment with increasing concentrations of Dox.

Reviewer #1 (Recommendations for the authors):

[…]

I don't disagree with the data interpretations but am having a hard time visualizing the biology of the system when PTCH is expressed because of the way the MBCD results are presented. The gist is that there is less free cholesterol in the outer membrane when PTCH is around, so MBCD doesn't have much to extract and PFO TIRF is not redistributing. However, since the lines don't shift following MBCD (Figure 2C as an example), it looks like cholesterol is locked in the membrane by PTCH, rather than already having been depleted. I think this is a visualization problem because everything is normalized to 1. Are the baseline readings significantly different between the samples? Is baseline higher/lower when PTCH is expressed? I needed to sketch it out for myself, and I feel like this is too much work for the reader. Is there a way to convey the results so they're more intuitive? More cartoons, maybe? The cartoons provided are very helpful.

We apologize that we did not clearly present the rationale for the way in which the kinetics of outer leaflet cholesterol extraction by MβCD were analyzed and presented. Briefly, our goal in establishing the TIRFM assay was to measure cholesterol accessibility in the membrane outer leaflet without relying on steady-state binding of PFO- or ALO-based probes. The steady-state binding of such probes has been used by us (in our 2019 eLife paper) and others (Zhang et al., 2018), with conflicting results (see the first section of the Results). The source of the conflicting results may lie in the high level of non-specific protein and membrane binding of the PFOD4 variants used in the papers from other groups. These issues have been extensively discussed in a previous Scientific Correspondence published in eLife (Courtney et al., 2018), where it was concluded that accurately inferring cholesterol content in membrane leaflets using hydrophobic PFOD4 variants used in (Zhang et al., 2018) should be accompanied by caution. Most importantly, the probes used by these other groups are not selective for accessible or active cholesterol (see Supplementary Figure 1e-k in (Liu et al., 2017)) over total cholesterol and so cannot be used to measure accessible cholesterol changes in response to Hedgehog signaling.

Given the pitfalls of using steady-state probe binding as the sole measure of accessible cholesterol, we developed a time-resolved, kinetic assay to monitor the rate of outer leaflet cholesterol extraction by MβCD. This assay is based on a decade of both experimental and theoretical work by many labs that are considered pioneers in membrane biology: McConnell, Lange, Steck, Slotte and others (Lange et al., 2004; Litz et al., 2016; McConnell and Radhakrishnan, 2003; Ohvo and Slotte, 1996; Radhakrishnan and McConnell, 2000). Indeed, careful measurement of the rates of cholesterol extraction by MβCD (not steady-state measurements of cholesterol content) first led to discovery of accessible cholesterol (summarized in (Lange and Steck, 2020)).

Kinetic assays are generally both more sensitive and less susceptible to artefacts. For example, the steady-state binding of PFOD4 to the inner leaflet is influenced by many factors other than the cholesterol content, such as the amount of PFOD4 expressed in an individual cell (noted by Reviewer #2) and the non-specific affinity of the probe for membranes. In contrast, cholesterol extraction by MβCD follows simple first-order, exponential decay kinetics: the half-life (t1/2) for extraction is independent of the starting baseline value (as it is for radioactive decay). In all the data presented in our paper, the curves represent the combined results from >20 individual cells (not single cells). Each of these cells shows a different baseline level of PFOD4H fluorescence at the membrane, likely because each cell expresses different amounts of the probe. This wide distribution in baseline values (Figure 1B) reduces sensitivity (e.g. while the median baseline values are different with and without PTCH1, the distributions overlap). We felt that the best way to analyze the data was to focus on the kinetics (or half-life of extraction) and to normalize the fluorescence for each cell to its own baseline value (set to 1) before averaging over all the cells analyzed. A classical paper on measuring accessible cholesterol in intact cells by Rothblat and colleagues (on which our assay is based) used this same analysis strategy (see Figures 1-4 in (Yancey et al., 1996)).

To show the impact of this normalization procedure, we now show data from our TIRFM assay analyzed in two different ways for select panels in Figures 2 and 3. In addition to baseline normalized data (Figures 2E-2F and Figures 3C, 3I), we provide panels (Figure 2—figure supplement 1C-1D and Figure 3—figure supplement 2A-2B) where the data is presented without any normalization to the baseline, with the raw fluorescence values (corrected for photobleaching) simply averaged at each time point over the ~20 cells analyzed. As this reviewer predicts and as shown by Figure 1B of the manuscript, the baseline values are indeed different: PTCH1 expressing cells start out with a higher level of PFOD4H binding to the inner leaflet. While the main conclusions from either representation are the same, it is much more difficult to compare the exponential decay phases (which are the focus of this assay) when the baselines are different. This is the reason we chose to represent the data as “fractional fluorescence remaining” relative to a value of “1” at baseline (Fluorescence at time t divided by Fluorescence at time zero).

Finally, we completely understand the statement that cholesterol looks “frozen” when PTCH1 is expressed. To address this issue we performed a dose-response curve using a range of MβCD concentrations in Figures 3D-3F of the manuscript. This experiment shows that cholesterol can still be extracted from PTCH1 containing membranes at higher concentrations of MβCD.

Re: Figure 6 and related to the point above: If PTCH is flipping cholesterol to the inner membrane, this should be reflected by PFO TIRF even in the absence of MBCD. Do you see a higher baseline in response to Dox induction of PTCH expression? The results presented in Figure 6 are less convincing that the rest of the figures, so it felt like the manuscript was ending with an afterthought. This point would be improved by additional experimental support and more discussion. There is only one sentence discussing the results in Figures 6B and C.

Thank you for this very helpful comment, which has prompted us to reorganize of the paper to address both the comments from Reviewer #1. As described in detail above in our response to Comment 1.1, we agree that the measurements using steady-state binding of probes are less sensitive and have a smaller dynamic range than measurements using the half-life of cholesterol extraction by MβCD. As this reviewer comment perceptively notes, this point is strikingly demonstrated when comparing the data shown in Figure 1 (steady-state binding) and the kinetic data shown in the rest of the manuscript. This is the reason our manuscript uses these kinetic measurements (rather than steady-state probe binding) to assess cholesterol accessibility in the membrane outer leaflet. As shown in Figure 1B and Figure 3—figure supplement 2A, we indeed see a higher baseline PFOD4H fluorescence at the inner leaflet when PTCH1 is expressed.

Prompted by this constructive comment, we have inverted the organization of the paper. We now begin by showing data on the effect of PTCH1 on the steady-state binding of TdTomato-PFOD4H or GFP-GRAM1b to the inner membrane leaflet (Figures 1B and 1C) and mNeon-ALOD4 to the outer leaflet (Figure 1—figure supplement 1A). Once the reader is exposed to changes in baseline fluorescence caused by PTCH1, we then introduce the rationale for the kinetic assay used in the rest of the manuscript. We carefully describe the limitations of the steady-state binding assays and also explain the normalization process for the kinetic assays (lines 125-138). In addition to baseline normalized data (Figures 2E-2F and Figures 3C, 3I), we provide panels (Figure 2—figure supplement 1C-1D and Figure 3—figure supplement 2A-2B) where the data is presented without any normalization to the baseline, with the raw fluorescence values (corrected for photobleaching) simply averaged at each time point over the ~20 cells analyzed.

We thank the reviewer for this comment as the reorganized paper is more logical in its progression.

Reviewer #2 (Recommendations for the authors):

Recent publications suggest that PTCH1 is a cholesterol transporter that mobilises cholesterol or a cholesterol derivative from the inner leaflet of the plasma membrane to the outer leaflet, where its concentration is about 10-fold higher (Zhang et al., 2018, Cell 175, 1352-1364). This transport against a concentration gradient requires energy, and previous literature indicates that sodium is the cation that provides such energy (Myers et al., Proc Natl Acad Sci U S A 2017;114(52):E11141-E11150).

Related question – Conceptual concern: why would PTCH1 require energy provided by a cation gradient to move cholesterol in favour of its concentration gradient? The related cholesterol transporter NPC1 transports cholesterol from the outer to inner leaflet without an energy gradient, and it lacks the acidic triad essential for PTCH1 activity.

The mention of a cholesterol gradient between the outer and inner leaflets is raised several times in this review, including the related statement that “the inner leaflet cholesterol is 1%-3%.” The question of the transbilayer distribution of cholesterol in the plasma membrane is a controversial and unresolved issue. Work from multiple groups using diverse approaches supports a roughly equivalent (50-50) distribution of cholesterol in the two leaflets (these studies are nicely summarized in Table 1 of (Steck and Lange, 2018)). Moreover, these studies also established that the concentration of cholesterol in the plasma membrane is ~30-40 mole%.

In contrast, work from one group using PFO-based probes modified to markedly increase their hydrophobicity supports the notion that the concentration of cholesterol in the outer leaflet is 10-fold higher than the inner leaflet (Buwaneka et al., 2021; Liu et al., 2017). It is worth noting that two published commentaries by independent experts in the field (one published in eLife) have challenged these conclusions (Courtney et al., 2018; Steck and Lange, 2018). Importantly, the eLife paper provided experimental evidence that the PFO-based probes used by the Cho group (and which were also used by the papers that our work is compared to in these reviews) cannot be used to reliably infer cholesterol abundances in the two leaflets because of non-specific membrane and protein binding. Courtney and colleagues also show that the inner leaflet cannot contain only 1%-3% cholesterol because a PFOD4 based probe that binds membranes only above a cholesterol mol% of >35% readily binds to the inner leaflet (Courtney et al., 2018). In addition, PFOD4H binds to membranes at cholesterol concentrations greater than 30%, not 1-3% (Johnson et al., 2012). Yet, we (Figure 2C in the manuscript) and others (Abe and Kobayashi, 2021; Maekawa and Fairn, 2015) readily detect its binding to the inner leaflet. As we note in lines 368-382 of the Discussion our work does not depend on (and does not inform) the current unresolved debates around the transbilayer distribution of cholesterol. However, given the uncertainty around the transbilayer distribution of cholesterol, we cannot make any statements about whether PTCH1 is moving cholesterol up or down its concentration gradient.

Second, we note that the use of sodium as the cation that provides energy is also uncertain. In contrast to the work implicating a sodium gradient referenced by this reviewer (Myers et al., 2017), work from the Salic lab has implicated the potassium gradient in driving PTCH1 function (Petrov et al., 2020). Our direct assays for PTCH1 activity support a role for the potassium gradient, in agreement with the Salic work. It is reassuring that both our work and the Salic work converged on a potassium gradient despite using completely different assays for PTCH1 activity.

PTCH1 contains an acidic triad (EED), conserved in bacterial RND permeases, that is essential for its activity as Smoothened repressor and likely allows co-transport of the cation.

In this study, Kinnebrew et al., follow their previous finding that indicates that sphingomyelin (SM) depletion potentiates Hedgehog signalling (Kinnebrew et al., eLife. 2019 Oct 30;8:e50051). SM localises in the outer leaflet of the plasma membrane and forms a complex with cholesterol, reducing availability of "free" or chemically-active cholesterol. Given that this suggests that increases in cholesterol concentration in the outer leaflet drives Smoothened activation, as opposed to the findings of the Beachy Lab, they re-evaluate PTCH1's transport directionality in the current study using total internal reflection fluorescence microscopy (TIRFM) to image reduction of binding of a cholesterol sensor domain fused to a fluorescent protein (TdTomato) to the inner leaflet when cholesterol is extracted from the outer lamella using methyl-β-cyclodextrine. The findings support the author's conclusion that PTCH1 reduces accessible outer leaflet cholesterol, but several methodological and conceptual questions remain:

– MβCD pinches accessible cholesterol out of the membrane, but it is unclear if the rate of extraction is comparable to the unknown rate of transport by PTCH1, or if a rate-limiting aspect of the model confounds the interpretation of the rate of PFOD4H-TdTomato fluorescence reduction. Therefore, while the assay can be optimal to capture spontaneous flip-flopping, it may not be a faithful readout of a facilitated transport event.

We agree that the rate of cholesterol extraction by MβCD cannot be used as a direct measure of the rate of cholesterol transport by PTCH1 (and are careful not to state this anywhere in the manuscript). What we can say is that the accessibility of outer leaflet cholesterol in membranes expressing PTCH1 is lower based on MβCD extraction rates. This rate can still be used to infer PTCH1 activity because is affected by the inactivation of PTCH1 using three independent strategies: a classical point mutation (D513Y, Figure 5), addition of its known inactivating ligand SHH (Figure 3) or dissipation of the K+ gradient (Figure 6). Finally, we have used two positive controls (Figures 2E and 2F) to show that the extraction rate is sensitive to the abundance of accessible cholesterol in the membrane. In conclusion, while the rate of cholesterol extraction by MβCD cannot be used to measure the rate of cholesterol transport by PTCH1, it can be used as a measure of accessible cholesterol in the outer leaflet (the way it is used in the manuscript).

– PFOD4H-TdTomato fluorescence in the steady-state is more likely to represent the inner leaflet cholesterol content. Because the TIRFM signals are normalised to t=0, this crucial information is not available in most experiments other than in Figure 6. This would be particularly useful in cells expressing PTCH1 and PTCH1-DL2, and after a few minutes of SHH addition, as the affinity of PFOD4H for cholesterol is close to the reported inner leaflet cholesterol level (~ 1-3 mol%).

This point has been discussed thoroughly in our response to comments 1.1, 1.2 and 2.2 above. We have outlined the shortcomings of using steady-state binding of PFO-based probes (especially those used in the prior papers from other groups) to infer cholesterol content in each leaflet and highlighted that our use of MβCD extraction kinetics represents a completely orthogonal measure of outer leaflet cholesterol accessibility. Importantly, our kinetic assay measures cholesterol accessibility in the outer leaflet (where sphingomyelin is located), not the inner leaflet.

As noted in our response to comment 2.1 above, the inner leaflet is unlikely to contain 1-3 mol% cholesterol. PFOD4H binds to membranes at cholesterol concentrations greater than 30%, not 1%-3% (Johnson et al., 2012). Yet we (Figure 2C in the manuscript) and others (Abe and Kobayashi, 2021; Maekawa and Fairn, 2015) readily detect its binding to the inner leaflet.

In response to this request, we now show data from our TIRFM assay normalized in two different ways for select panels in Figures 2 and 3. In addition to baseline normalized data (Figures 2E-2F and Figures 3C, 3I), we provide panels (Figure 2—figure supplement 1C-1D and Figure 3—figure supplement 2A-2B) where the data is presented without any normalization to the baseline, with the raw fluorescence values (corrected for photobleaching) simply averaged at each time point over the ~20 cells analyzed.

– If myriocin treatment increases accessible cholesterol in the outer leaflet, wouldn't it increase the inner leaflet cholesterol content by flip-flop? If that is the case, the non-normalised TIRF signal should be higher at t=0 than in vehicle-treated cells.

Yes, as we show in Figure 2-figure supplement 1B, myriocin treatment leads to increased steady-state binding of the PFOD4 probe to the inner leaflet of the plasma membrane.

– The concentration of SHH ligand used to module cholesterol availability seems excessively high (10-6 M). The same group used 25 nM in the previous study as a "high, saturating SHH concentration", in line with most groups using SHH in the range of 50-100 nM. A dose-response effect will also inform if the IC50 of SHH in the TIRF assay agrees with its Kd for PTCH1 and the EC50 for stimulation of Gli-dependent transcription.

– The lipid modifications of SHH play a key role in the asymmetric binding mode to PTCH1 dimers. The article does not detail the modifications of the SHH used.

– Induction of PTCH1 expression increases absolute TIRF signals in Figure 6, suggesting higher inner leaflet cholesterol. However, this interpretation depends on equal total fluorescence, i.e. equal expression of the fluorescent sensor proteins regardless of PTCH1 expression and SHH signalling.

– If PTCH1 reduces accessible cholesterol in the outer lamella, one would expect to observe a reduction in binding of an extracellular cholesterol sensor, equivalent to the PFOD4H-TdTomato.

– The biggest limitation of this study is the reliance on overexpressed PTCH1. The effect of SHH addition to Hh-competent cells expressing endogenous PTCH1 (such as NIH 3T3 cells) on cholesterol sensor measurements is essential, even if it cannot be determined in the primary cilium membrane.

– This study clashes with previous reports using confocal microscopy vs TIRF. One question that comes to mind is if a different conclusion would be drawn using the PFOD4H-TdTOmato sensor in standard confocal imaging to image changes in lateral membranes that are more readily exposed to solvent and SHH than the basal membrane attached to the substratum.

However, I identified some areas that need to be addressed. I would like you to consider providing additional evidence for some of my key concerns:

1 – Provide baseline absolute levels of fluorescence in each condition.

This point has been discussed thoroughly in our response to comments 1.1 and 1.2 above. We again note that the purpose of our assay is to measure the outer leaflet accessibility of cholesterol without using steady-state binding of PFO-based probes, which has been done before and yielded conflicting results (Kinnebrew et al., 2019; Zhang et al., 2018). In addition, the half-life of MβCD-mediated cholesterol extraction (the parameter used to infer cholesterol accessibility in our manuscript) is independent of the starting baseline fluorescence value given the simple exponential decay kinetics of the extraction reaction.

In response to this request, we now show data from our TIRFM assay normalized in two different ways for select panels in Figures 2 and 3. In addition to baseline normalized data (Figures 2E-2F and Figures 3C, 3I), we provide panels (Figure 2—figure supplement 1C-1D and Figure 3—figure supplement 2A-2B) where the data is presented without any normalization to the baseline, with the raw fluorescence values (corrected for photobleaching) simply averaged at each time point over the ~20 cells analyzed.

2 – Demonstrate that induction of PTCH1 does not affect total expression of PFOD4H-TdTomato.

A data panel showing that TdTomato-PFOD4H abundance does not change when PTCH1 is induced with Dox is provided in Figure 3—figure supplement 1A.

As we discuss in the response to Comment 1.1 and in the Results section of the revised manuscript (lines 125-128), this effect is relevant only for the steady-state measurements shown in Figure 1. The half-life of cholesterol extraction by MβCD follows exponential decay kinetics and is hence independent of the starting baseline fluorescence value. This feature is a major strength of our kinetic assay since it allows measurement of outer leaflet cholesterol accessibility in a manner independent of probe expression in cells. Finally, the reversibility of the PTCH1 effect with acute SHH exposure makes it unlikely that our results are due to changes in PFOD4H expression.

3 – Investigate the effect of adding more physiological concentrations of Shh (in the 10-50 nM range) to Hh-competent cells expressing the cholesterol sensor. Potential controls of acute silencing of PTCH1.

We used a higher concentration of SHH in this manuscript because we are working in a system in which PTCH1 is overexpressed. As a control to ensure that SHH at this concentration does not have off-target effects, we used (Figure 3) a widely-studied mutant of PTCH1 that cannot bind to SHH (PTCH1-ᐃL2). In animals ranging from flies to humans PTCH1-ᐃL2 fails to bind or respond to SHH but retains its ability to suppress SMO activity (Briscoe et al., 2001). SHH (even at the higher concentration used in our assays) has no effect on cells expressing PTCH1-ᐃL2, showing that its effects in our system are specific.

There is considerable evidence (both from our experience and from the published literature) that increasing PTCH1 expression increases the amount of SHH that must be added to overcome the inhibitory effect of PTCH1 on Hh signaling.

In response to this comment we have provided a SHH dose-response curve for our TIRFM assay in Figure 3G. The EC50 (concentration of SHH that causes a half-maximum increase in cholesterol accessibility) is ~160 nanomolar.

With respect to PTCH1, we are already using a Dox-inducible system so that in the same cell line we can assess outer leaflet cholesterol accessibility in the absence (-Dox) or presence (+Dox) of PTCH1. In addition, we inactivate PTCH1 acutely by adding SHH or dissipating the K+ gradient. Silencing PTCH1 by withdrawing Dox is possible but will not be acute due to the long half-life of the protein and latency of Dox washout.

4 – Testing changes in binding of an extracellular cholesterol sensor in the system.

We have attempted this experiment using recombinant PFOD4-GFP or ALOD4-GFP added to the extracellular medium. However, we cannot use an extracellular probe to monitor the kinetics of cholesterol extraction by extracellular MβCD using TIRFM. The >50 kDa PFOD4-GFP and ALOD4-GFP probes have poor and nonuniform access to the space between the cell and the coverslip, precluding the use of TIRFM, which is crucial for our time-resolved, kinetic assay (see Discussion under comment 2.10 below).

However, we have used steady-state binding of extracellular PFO* in our 2019 eLife paper to show that SHH causes an increase in cholesterol accessibility in the ciliary membrane of NIH/3T3 cells (a result in complete agreement with the conclusions of our current manuscript). As requested in this comment, we used flow cytometry to measure the binding of an extracellular cholesterol sensor (mNeonGreen-ALOD4) to the outer leaflet of the PTCH1 expressing HEK293T cells used for the kinetic studies throughout this manuscript (Figure 1—figure supplement 1). As with other steady-state measurements, the changes are small; however, they are consistent and support the model that PTCH1 expression (in a SHH-reversible manner) decreases outer leaflet cholesterol accessibility. Differences in the FACS-based binding assay may also be dampened because it requires detachment of cells from the tissue culture plate and solution staining prior to flow cytometry. Again, we emphasize that the kinetic assays based on MβCD extraction reveal much more significant differences spread over a larger dynamic range compared to steady-state binding measurements.

5 – Indicate source and molecular details of the SHH ligand used: is it lipidated, is the Ile-Ile mimic of the N-terminal palmitate? Please add this to the methods.

If Shh is unmodified, could you test if dual lipidated SHH has the same effects at a lower concentration?

We apologize for not making this more clear in the methods. We are indeed using SHH carrying a double Ile at its N-terminus (known as SHH-C24II) to mimic the palmitate attached to native SHH. This protein has been widely used in the literature, including by us in our 2019 eLife paper and in all other papers on Hedgehog signaling we have published over the last 7 years (Kinnebrew et al., 2019). Pepinsky and colleagues first developed the SHH-C24II variant in a seminal paper over two decades ago and validated its use as an easy to purify and well-behaved substitute for the endogenous ligand (Taylor et al., 2001). Full details of the SHHC24II used in our manuscript are now provided in the Methods (lines 638-645).

– The contradictory findings are unlikely to be explained simply by a difference in the cholesterol sensor used (PFO-D4H-tdTOmato vs PFO-D4H tagged with a small solvatochromic fluorophore). Additional controls and a deeper discussion of the geometry of the assay and the potential impact of measuring MbetaCD-induces changes in fluorescence vs steady-state levels will be necessary to understand the differences.

Also: 6 – I'd also like to see a deeper discussion on the potential explanations of the Zhang et al., results. You are creating a lively controversy in the field and would strongly benefit from trying to figure out why your conclusions are correct and the other is not when the systems are so similar in most ways.

The paper from the other group referred to in this comment did not use PFOD4H as a probe of inner leaflet cholesterol, but rather a triple mutant of PFOD4 (Y415A/D434W/A463W) coupled to a solvatochromic fluorophore which was produced as a recombinant protein and microinjected into cells (Liu et al., 2017; Zhang et al., 2020, 2018). This probe is considerably more hydrophobic than PFOD4H. An independent ELife paper has highlighted the flaws in these probes-- they show a high level of non-specific protein and membrane binding and hence do not accurately reflect membrane cholesterol content (Courtney et al., 2018). In addition, the authors themselves have shown that these probes cannot be used to measure accessible cholesterol in membranes (Supplementary Figure 1ek of (Liu et al., 2017)) and hence cannot be used to comment on changes in accessible cholesterol in response to SHH.

The assays used by our current manuscript and the work from other groups described in this comment are fundamentally different. Work from others used the steady-state inner leaflet binding of an highly engineered PFOD4H variant that does not selectively detect accessible cholesterol (shown in their own manuscript (Liu et al., 2017)) and hence has lost the critical property that makes it useful as a probe of how SHH/PTCH1 impact accessible cholesterol. In contrast, we have developed a time-resolved, kinetic assay to monitor the rate of outer leaflet cholesterol extraction by MβCD, a sensitive indicator of accessible cholesterol in cells (Haynes et al., 2000; Yancey et al., 1996). Our measurement of accessible cholesterol does not depend on steady-state probe binding (which has produced conflicting results), but rather on measuring the rate of cholesterol transfer to MβCD, an assay which has no similarity to the one used by other groups.

In response to this comment, we have significantly expanded our discussion of the differences between our manuscript and the work from others outlined above. We begin the Results with a section entitled Conflicting models for the influence of PTCH1 on membrane cholesterol organization (lines 43-93). In addition, we have also added a section in the Discussion section entitled Comparison to alternative models for PTCH1 transporter function (lines 331-353). We appreciate this suggestion as it provides a better context for our manuscript relative to the major competing models for the function of PTCH1 in cholesterol transport.

https://doi.org/10.7554/eLife.70504.sa2

Article and author information

Author details

  1. Maia Kinnebrew

    Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7344-8231
  2. Giovanni Luchetti

    1. Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    2. Department of Physiological Chemistry, Genentech, South San Francisco, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Ria Sircar

    Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    Contribution
    Investigation, Methodology, Resources
    Competing interests
    No competing interests declared
  4. Sara Frigui

    Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  5. Lucrezia Vittoria Viti

    Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Tomoki Naito

    Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
    Contribution
    Methodology, Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8393-3601
  7. Francis Beckert

    Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    Contribution
    Methodology, Software
    Competing interests
    No competing interests declared
  8. Yasunori Saheki

    Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
    Contribution
    Methodology, Resources, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1229-6668
  9. Christian Siebold

    Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
    Contribution
    Formal analysis, Methodology, Resources, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6635-3621
  10. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Methodology, Resources, Supervision
    Competing interests
    is a reviewing editor for eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336
  11. Rajat Rohatgi

    Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States
    Contribution
    Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - original draft, Writing – review and editing
    For correspondence
    rrohatgi@stanford.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

Funding

Cancer Research UK (C20724)

  • Christian Siebold

Cancer Research UK (A26752)

  • Christian Siebold

European Research Council (647278)

  • Christian Siebold

National Institutes of Health (GM118082)

  • Rajat Rohatgi

National Institutes of Health (GM106078)

  • Rajat Rohatgi

National Institutes of Health (HL20948)

  • Arun Radhakrishnan

Welch Foundation (I-1793)

  • Arun Radhakrishnan

Leducq Foundation (19CVD04)

  • Arun Radhakrishnan

Ministry of Education, Singapore (MOE2017-T2-2-001)

  • Yasunori Saheki

Ministry of Education, Singapore (MOE-T2EP30120-0002)

  • Yasunori Saheki

National Science Foundation (Predoctoral Fellowship)

  • Maia Kinnebrew

Ford Foundation (Predoctoral Fellowship)

  • Giovanni Luchetti

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

Acknowledgements

The authors thank Chandni Patel and Hermann Broder Schmidt for help on experiments (Figure 6) to alter transmembrane ion gradients, Greg Fairn for providing the TdTomato-PFOD4H probe and Kristen Johnson, Ted Steck, and Yvonne Lange for helpful discussions.

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Duojia Pan, UT Southwestern Medical Center and HHMI, United States

Publication history

  1. Received: May 27, 2021
  2. Accepted: October 25, 2021
  3. Accepted Manuscript published: October 26, 2021 (version 1)
  4. Version of Record published: December 8, 2021 (version 2)

Copyright

© 2021, Kinnebrew et al.

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.

Metrics

  • 1,386
    Page views
  • 362
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Maia Kinnebrew
  2. Giovanni Luchetti
  3. Ria Sircar
  4. Sara Frigui
  5. Lucrezia Vittoria Viti
  6. Tomoki Naito
  7. Francis Beckert
  8. Yasunori Saheki
  9. Christian Siebold
  10. Arun Radhakrishnan
  11. Rajat Rohatgi
(2021)
Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
eLife 10:e70504.
https://doi.org/10.7554/eLife.70504

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Haikel Dridi et al.
    Research Article Updated

    Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in Caenorhabditis elegans (C. elegans); however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in ‘leaky’ channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2–3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.

    1. Cell Biology
    Desiree Schatton et al.
    Research Article

    Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.