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Comment on ‘Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol’

  1. Kevin C Courtney
  2. Karen YY Fung
  3. Frederick R Maxfield
  4. Gregory D Fairn  Is a corresponding author
  5. Xiaohui Zha  Is a corresponding author
  1. University of Ottawa, Canada
  2. St. Michael’s Hospital, Canada
  3. Weill Cornell Medical College, United States
  4. Ottawa Hospital Research Institute, Canada
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Cite this article as: eLife 2018;7:e38493 doi: 10.7554/eLife.38493

Abstract

The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (Liu et al., 2017). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.

https://doi.org/10.7554/eLife.38493.001

Introduction

Cholesterol is an essential molecule in mammalian cells as it supports several critical functions of the plasma membrane and other organelles. The majority of studies have reported that cholesterol constitutes 35–40 mol% of the plasmalemmal lipids (van Meer et al., 2008), and most studies support the notion that there is more cholesterol in the cytoplasmic leaflet than in the exofacial leaflet, or that the balance is close to even (Kobayashi and Menon, 2018; Steck and Lange, 2018). It was surprising, therefore, when Liu et al. reported that the abundance of cholesterol in the exofacial leaflet is about an order of magnitude higher than that in the cytoplasmic leaflet, and that the plasmalemmal cholesterol content is 22–23 mol%. Their conclusions, particularly the cholesterol transbilayer distribution, were based on a series of D4 mutants that require different minimum concentrations, or thresholds, of cholesterol in the membrane to bind liposomes in vitro. However, such thresholds may not depend on cholesterol concentrations alone. Phospholipids that surround cholesterol could influence the accessibility of the D4 probes to cholesterol. Given the complexity of the phospholipid compositions in the plasma membrane and the importance of understanding cholesterol distribution in the plasma membrane, we decided to put these D4 probes, as used by Liu et al., through more rigorous tests. The results here challenge the applicability of this method to quantitatively measure the transbilayer distribution of cholesterol in live cells.

Results

Phospholipid head groups impact DAN-D4 binding 

For cholesterol-binding, perfringolysin O (PFO) and its derivatives require a minimum cholesterol concentration, or threshold, in the membrane to bind. Such a threshold is known to be strongly influenced by the membrane phospholipid composition (Flanagan et al., 2009; Nelson et al., 2008; Sokolov and Radhakrishnan, 2010). Although Liu et al. stated that D4 binding is unaffected by phospholipid composition, others have reported that it is sensitive to both the acyl chain composition and the phospholipid head group (He et al., 2017; Maekawa and Fairn, 2015a).

Specifically, Liu et al. used a defined liposome (POPC/POPS/cholesterol) and several D4 variants, labeled with acrylodan (DAN) or NR3, to generate a series of thresholds covering a range of cholesterol concentrations (Figure 1b & c in Liu et al., 2017). They then claimed that such thresholds hold true for: (1) DAN-D4 and DAN-D434A for exofacial leaflet mimic (PC/SM) liposomes (Supplementary Figure 1e-h, in Liu et al., 2017); and (2) for NR3-YDA and NR3-QYDA for cytoplasmic leaflet mimic (PC/PE/PS/PI) liposomes (Supplementary Figure 1j-k, in Liu et al., 2017). These thresholds were then applied to interpret D4 variants’ binding to the plasma membrane (Figure 1d & f in Liu et al., 2017). To test if this approach is valid, we examined whether DAN-D4 could similarly bind membranes that are as different as the exofacial and cytoplasmic leaflet of the plasma membrane but with identical cholesterol concentrations. For this, we generated liposomes that roughly mimic the exofacial (POPC/egg SM/cholesterol, 36:24:40) or the cytoplasmic leaflets (POPC/POPE/POPS/soy PI/cholesterol, 18:18:18:6:40) of the plasma membrane.

D4 binding is influenced by phospholipid composition and is subject to competition from proteins.

(A) Purified DAN-D4 (0.5 µM) was incubated with 100 µM large unilamellar vesicles (LUVs) composed of POPC/egg SM/cholesterol (36:24:40) and POPC/POPE/POPS/soy PI/cholesterol (18:18:18:6:40). The change in fluorescence emission (ΔF) at 450 nm is used to approximate cholesterol-dependent liposome binding and is corrected for non-specific binding to a cholesterol-free liposome. The results were normalized to the maximal ΔF (ΔFmax). (B) DAN-D4 (0.5 µM) binding to increasing concentrations of phosphatidylcholine/cholesterol (60:40) LUVs with various phosphatidylcholine acyl chain saturation. (C) DAN-D4 (0.5 µM) binding to 100 µM DOPC/cholesterol (60:40) LUVs in the presence of increasing concentrations of rat liver cytosol. The change in fluorescence was determined relative to cholesterol-free liposomes at 450 nm and then normalized to the control (ΔF/F). All data were acquired with a PTI scanning spectrofluorometer (ex. 380 nm and em. 420–560 nm). Each experiment was repeated at least three times and error bars represent standard error of the mean.

https://doi.org/10.7554/eLife.38493.002

Using identical probes and methodologies to those used by Liu et al., we isolated recombinant D4 from E. coli, conjugated the proteins with the solvatochromic dye, DAN, and repeated the in vitro binding experiments as performed by Liu et al. As depicted in Figure 1A, for liposomes with constant cholesterol concentration (40%), DAN-D4 preferred the mimic of exofacial leaflet (PC/SM) to the mimic of cytoplasmic leaflet (PC/PE/PS/PI); the amount of D4-DAN that binds the PC/SM liposomes is more than double that which binds PC/PE/PS/PI liposomes. Thus, it is evident that the threshold for D4 is not identical in liposomes that mimic exofacial and cytoplasmic leaflets of the plasma membrane. Varied DAN-D4 binding in vivo cannot, therefore, be directly interpreted as cholesterol concentration in the plasma membrane. In addition, the phospholipids in the plasma membrane are far more complex than these liposomes, which further challenges the use of these thresholds for calibration as used by Liu et al.

Acyl chain saturation of phospholipids impacts DAN-D4 binding 

It is known that D4 binding to cholesterol in the membrane depends on the degree of cholesterol exposure in that membrane, as a consequence of cholesterol interactions with surrounding phospholipids (He et al., 2017; Maekawa and Fairn, 2015a). Both head group and acyl chain length impact D4 binding. However, it is the acyl chain saturation, that is, the number of double bonds, that most significantly influences D4 binding (He et al., 2017; Maekawa and Fairn, 2015a). We therefore sought to test the effect of acyl chain saturation on DAN-D4 binding. To do so, we again used liposomes with constant cholesterol (40 mol%) but varying acyl chain saturation. As depicted in Figure 1B, relative to phospholipid with no double bonds in the acyl chain (DPPC, 16:0,16:0), the introduction of a single double bond (POPC, 16:0, 18:1) significantly enhanced the binding of the DAN-D4 to the liposomes. The DAN-D4 binding was further elevated when two double bonds were introduced (DOPC, 18:1, 18:1). Although Liu et al. did study the effects of phospholipid composition on D4 binding (supplementary 1h and i), they did not systematically test the effect of acyl chain saturation in phospholipids, particularly those most abundant in the plasma membrane. Specifically, the lipids used by Liu et al. are primarily POPC and otherwise always contained one saturated and one unsaturated acyl chain. Nevertheless, our experiments clearly demonstrate that both acyl chain saturation and phospholipid head group significantly impact DAN-D4 binding to liposomes. The fact that DAN-D4 binding to the cytoplasmic leaflet-like liposomes was significantly attenuated, compared to those that mimic the exofacial leaflet, suggests that Liu et al. could have underestimated the cholesterol content in the cytoplasmic leaflet of plasma membrane. More importantly, as the lipid classes and species in live cells are significantly more complex than the liposomes we used here, it is unlikely that the liposome-based calibration, as in the approach employed by Liu et al., could be regarded as a true proxy for D4 binding to the exofacial and cytoplasmic leaflets of the plasma membrane. Quantitative interpretation of D4 binding in live cells is extremely difficult, if at all possible, even with multiple rigorous calibrations. Additionally, within cells, the cytoplasm is very rich in proteins, which could further complicate the binding of D4 to cholesterol (see below).

DAN-D4 binding is highly sensitive to proteins in the medium 

Liu et al. reported that microinjected D4 and the variants D4D434A and D4D434A, A463W, failed to bind to the cytoplasmic leaflet of the plasma membrane (Liu et al., 2017). This was their key evidence to conclude that there is little cholesterol in the cytoplasmic leaflet. This observation was surprising as it has been reported previously that both wild-type D4 and a D434S mutant (comparable to D434A) are capable of binding to the cytoplasmic leaflet of the plasma membrane (Maekawa and Fairn, 2015b; Abe et al., 2012). This raises the possibility that the microinjected DAN-D4 proteins were not behaving as expected. One of the potential confounders is the presence of cytosolic proteins, which would interfere with DAN-D4 binding to cholesterol in the cytoplasmic leaflet of the plasma membrane. DAN-D4 could bind cytosolic proteins, which would titrate away the microinjected DAN-D4 and prevent DAN-D4 from binding to the cytoplasmic leaflet of the plasma membrane. The protein concentration in cells is estimated to be about 100 mg/ml (Zeskind et al., 2007; Luby-Phelps, 2000). With such a high protein concentration, even weak affinity of DAN-D4 to cytosolic proteins could reduce the effective concentration available for binding to membranes. To examine the potential impact of proteins, we performed DAN-D4 binding experiments in vitro in the presence of rat liver cytosol (RLC). As depicted in Figure 1C, the inclusion of RLC in the binding assay reduced DAN-D4 binding to the liposomes in a dose-dependent manner with an almost complete ablation of binding at 1.5 mg/ml. Thus, the capability of microinjected DAN-D4 to bind the cytoplasmic leaflet of the plasma membrane could be severely diminished in live cells, regardless of cholesterol content in the membrane.

D4D434A and D4D434A,A463W are capable of binding the cytoplasmic leaflet of the plasma membrane in live cells 

mCherry-D4 and mCherry D4D434S have been shown previously to bind to the cytoplasmic leaflet of the plasma membrane. We thus sought to determine whether the new variants of the D4, used by Liu et al. (2017), could similarly bind the cytoplasmic leaflet of the plasma membrane. As shown in Figure 2A, exogenously expressed mCherry-D4D434A and mCherry-D4D434A,A463W, in fact, do bind to the cytoplasmic leaflet of the plasma membrane. Importantly, these mCherry-tagged probes were responsive to changes in cholesterol: they were displaced from the cytoplasmic leaflet following extraction of the plasmalemmal cholesterol by methyl-β-cyclodextrin (Figure 2B). Thus, the lack of binding of the DAN-D4 variants, as shown by Liu and colleagues, is not likely to result from insufficient cholesterol in the cytoplasmic leaflet. Noticeably, liberation of the mCherry-D4 variants from the PM following cholesterol extraction is accompanied by the appearance of bright puncta within the cytosol. This cannot be a result of a sudden increase in endomembrane cholesterol, as acute cholesterol extraction would only lower cellular cholesterol, including endomembranes. However, without cholesterol-rich membrane to bind, D4 could form aggregates within the cytoplasm or be bound to unidentified membrane structures.

D4 variants can bind to the cytoplasmic leaflet of the PM in a cholesterol-dependent manner.

(A) CHO cells transiently transfected with mCherry-D4D434A and D4D434A, A463W and the plasma membrane marker, Pleckstrin homology domain of phospholipase C δ (PH-PLC δ) were examined using spinning-disc confocal microscopy. (B) Live-cell images were acquired of cells expressing the same probes as in (A) following incubation with 10 mM methyl-β-cyclodextrin (mβCD) for 20 min to extract plasmalemmal cholesterol. Scale bar, 10 µm. (C) Quantitation of the plasmalemmal enrichment of the mCherry signal seen in (A) and (B). means ± std. dev. n = 20.

https://doi.org/10.7554/eLife.38493.003

Discussion

It would be significant in membrane biology and physiology if there was a 10-fold enrichment of cholesterol in the exofacial leaflet over that in the cytoplasmic leaflet of the plasma membrane in mammalian cells. In particular, Liu et al. (2017), concluded that generation and maintenance of such a 10-fold gradient would be an ATP-consuming process, incurring a huge energy demand as cholesterol can flip spontaneously in membranes with a t1/2<1 s (Steck and Lange, 2018). We sought to interrogate the use of the D4 probes to quantitatively determine their reliability for measuring cholesterol in the exofacial and cytoplasmic leaflets. Here we have repeated and extended vital control experiments and found that the data reported in the study by Liu et al. cannot be extrapolated to provide precise measurements of cholesterol, especially for the cytoplasmic leaflet of the plasma membrane.

Full-length PFO toxin and recombinant D4 have been studied extensively for their ability to bind cholesterol. It is known that neighboring phospholipids influence the ability of D4 to sense and bind to cholesterol. Because of its small hydroxyl head group, cholesterol has to be shielded from the aqueous environment by surrounding phospholipids. The degree of shielding is influenced by the lateral packing of lipids (i.e., both the acyl chain composition of the lipids as well as the structure of the head group). Thus, when included with lipids with unsaturated acyl chains, such as DOPC, cholesterol is more readily accessible by D4 than when in membranes with saturated DPPC (Figure 1B). This observation alone demonstrates that D4 recognizes accessible or chemically active cholesterol, not bulk or total cholesterol.

Still, why does microinjected D4 not bind to the plasma membrane when the heterologous expressed mCherry versions do? One possibility is that the addition of the lipophilic DAN or the Nile red derivative, as used by Liu and colleagues, may increase the affinity of D4 variants to cytosolic proteins. Our observation (Figure 1C) supports this possibility. mCherry is not likely to alter the affinity of D4 to cytosolic proteins. Regardless of the fluorophore attached to the D4, the sensors will have to compete with endogenous cholesterol-binding proteins for the accessible pool of cholesterol in membranes.

Genetically coded biosensors for phospholipids have been used by cell biologists for two decades. However, lipid and cholesterol sensors such as D4 must be interpreted with rigor. In particular, cholesterol partitioning between leaflets in the plasma membrane is even more complicated to assess by such binding, as cholesterol can spontaneously flip-flop between leaflets of a bilayer (Leventis and Silvius, 2001). Thus, the binding of cholesterol by an exogenous membrane-impermeant probe on the exofacial surface would likely trap cholesterol and alter the cholesterol distribution. This situation was elegantly illustrated in a recent paper, where binding ~1% of the cholesterol in the exofacial leaflet using a D4 homolog was sufficient to trigger a ‘lack of cholesterol’ signal on the ER membrane inside the cells (Infante and Radhakrishnan, 2017). Although D4 and its derivatives are useful in some focused studies to make endpoint measurements or inhibit cholesterol trafficking, we conclude that these tools cannot be used to assess cholesterol partitioning between two leaflets of plasma membranes in live cells.

Materials and methods

Recombinant protein production and liposomal binding

Domain 4 (amino acids 391–500) of PFO and its variants were provided by Liu et al. (2017), expressed as GST chimeric proteins using the pGEX-4T-1 vector transformed into BL21 E. coli as previously described. Following purification with glutathione-conjugated affinity resin (GE Healthcare) the recombinant proteins were covalently modified with acrylodan (6-acryloyl-2-dimethylaminonaphthalene) or simply ‘DAN’ (ThermoFisher) and liberated from the GST tag by incubation with thrombin protease, as described in Liu et al. (2017).

Large unilamellar vesicles were produced by first drying lipids in chloroform in glass tubes under a stream of nitrogen, followed by vacuum dessication for at least 1 hr. The lipids were resuspended in aqueous buffer and subjected to a freeze/thaw cycle before extrusion through 100 nm polycarbonate membrane. The binding of the DAN-D4 to the liposomes results in insertion of DAN into the hydrophobic bilayer that is accompanied by a shift in its emission spectra from a peak of ~490 nm to ~450 nm. The change in fluorescence emission (ΔF) at 450 nm is used to estimate cholesterol-dependent membrane binding, relative to identical but cholesterol-free LUVs. The results were normalized to the maximal ΔF (ΔFmax). To examine the effect of a lipid head group, liposomes were generated with the following compositions: exofacial leaflet-like POPC/egg SM/cholesterol (36:24:40) and cytoplasmic leaflet-like and POPC/POPE/POPS/soy PI/cholesterol (18:18:18:6:40). 100 µM liposomes were incubated with 0.5 µM DAN-D4 to determine relative D4 binding. Additionally, to examine the contribution of acyl chain composition, we compared the binding of the D4 to a series of PC/cholesterol (60:40) liposomes in which the molecular species of PC was dipalmitate, palmitate-oleate, or dioleate. DAN-D4 binding curves were generated by incubating 0.5 µM DAN-D4 with increasing liposome concentrations from 0.5 µM to 160 µM. For the effect of rat liver cytosol, DOPC/cholesterol (60:40) liposomes were generated in the same fashion as above but were initially resuspended in diluted rat liver cytosol at the indicated concentration. Again, 100 µM liposomes were incubated with 0.5 µM DAN-D4 in the presence of increasing rat liver cytosol. The change in fluorescence was determined relative to cholesterol-free liposomes at 450 nm and then normalized to the control (ΔF/F). Fluorescence measurements were aquired with a scanning spectrofluorometer (Photon Technologies International) (ex. 380 nm and em. 420–560 nm).

Fluorescence microscopy

The open-reading frames of D4D434A and D4D434A, A463W were subcloned into the pmCherry-C1 expression plasmid. Chinese hamster ovary (CHO) cells were maintained in DMEM media containing 10% fetal bovine serum. For imaging experiments, cells were seeded on 18 mm coverslips and transiently transfected with the indicated plasmids using X-tremeGENE9 (Roche) and returned to the incubator. The next day, live cells were transferred to a chamber slide and imaged using spinning-disc confocal microscopy. To determine the impact of cholesterol removal on D4 localization, cells were treated with 10 mM methyl-β-cyclodextrin for 20 min before imaging. The spinning-disc imaging system used is based on a Leica DMIRE2 equipped with a Yokogawa CSU X1 scan head and a 60 × (NA 1.35) oil immersion objective using a Hamamatsu C9100-13 electron-multiplying charge-coupled device (EM-CCD) camera. Excitation light was provided by 491 nm (50 mW) and 561 nm (50 mW) lasers, and emitted light was collected after passage through 515/40 and 594/40 nm emission filters. Post-acquisition analysis was conducted using the region of interest tool in ImageJ. Briefly, in highly magnified images regions of the plasma membrane (PM), cytosol, and outside the cell (background) were analyzed for mean fluorescence intensity. The plasma membrane enrichment was calculated using the background subtracted values and the following equation; (PM-Cyto) ÷ Cyto. The graph was generated using Prism (GraphPad) and includes the individual data points (n = 20) with the means ± the standard deviation indicated.

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Decision letter

  1. Arun Radhakrishnan
    Reviewing Editor; University of Texas Southwestern Medical Center, United States
  2. Philip A Cole
    Senior Editor; Harvard Medical School, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Comment on "Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol"” for consideration by eLife. Your article has been reviewed by Philip Cole as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Satyajit Mayor (Reviewer #1); Daniel Wüstner (Reviewer #3). A further reviewer remains anonymous.

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

Summary:

The authors challenge the conclusions made in a recently published paper regarding the distribution of cholesterol between the two leaflets of plasma membranes (PMs) (Liu et al., 2017). Using a domain from a cholesterol-binding bacterial protein, this earlier paper had reported that the outer leaflet of the PM is ~12-fold enriched in cholesterol compared to the inner leaflet, a finding that is at odds with many published studies that report a roughly even distribution of cholesterol between the two PM leaflets. The current "Comment" submission raises several valid concerns regarding the sensor used by Liu et al., and how that may have skewed the analysis in their study. The subject of trans-bilayer cholesterol distribution has received renewed attention in light of its significance in understanding cellular cholesterol transport (mediated by NPC1 and other proteins) and cholesterol-mediated cellular signaling by the Hedgehog signaling pathway. The reviewers agreed that this "Comment" challenging the methodology and analysis used by Liu et al., would serve not just as a cautionary critique but provide a timely and clarifying contribution to this field.

Essential revisions:

To make this Comment article as solid as possible, the reviewers request the following essential revisions:

1) The authors show the effect of composition on DAN-D4 binding at a single concentration of D4 (which incidentally is not mentioned in the text). It would be desirable that a binding isotherm be performed/shown that indicates that they are achieving at saturation levels for all compositions tested. It is not clear whether it is the 'availability/accessibility' of cholesterol that is changing or the Kd of D4 for the cholesterol in the membrane. Coupled with the lack of information about the concentrations of the D4 protein, it is hard to come to a firm conclusion.

2) The authors challenge the lack of binding of the DAN- and NR3-labeled D4s to the inner PM leaflet in the Liu et al. study by showing that heterologously expressed mCherry D4s do label the inner leaflet. The impact of this new data piece is modest: transiently transfected fluorescent fusions of D4 probes have been previously reported to localize to the inner plasma membrane leaflet and with the substantial cell-to-cell variation in the labeling pattern observed with transient expression, single cell exemplary images are questionable. The authors suggest that lack of detection of the NR3-D4 at the inner leaflet may be due to low amounts of D4s being microinjected into cells or due to the presence of competing endogenous proteins (the authors use BSA as a case study to make this point). The authors' discussion of this point is not compelling since transient transfection may be even more difficult to control than microinjection in terms of protein dosage and it is not apparent why the transiently expressed protein would behave more correctly. This should be quantitatively addressed by comparing their binding isotherms – it is possible that mCherry D4 variants behave differently from both NR3-D4 variants or the DAN D4 variants.

3) The authors need to be cautious in their interpretations regarding point 2 – the exoplasmic leaflet binding may also be subject to interference by proteins.

4) Liu et al., did test liposomes with substantial amounts of a typical inner leaflet lipid (20 mol% PS, in addition to PC and cholesterol) and also PC/PE/PS/PI containing liposomes, but not systematically with all D4 variants. The authors here are more systematic and additionally show that the acyl chain composition of PC affects D4 binding. They make a valid point but should not mischaracterize Liu et al.

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

Thank you for resubmitting your work entitled "Comment on "Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol”" for further consideration at eLife. Your revised article has been favorably evaluated by a Senior Editor (Philip Cole) and a Reviewing Editor (Arun Radhakrishnan).

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Please make the following clarifying revisions.

1) Results section: Please include an additional reference to the large body of earlier work that also suggests ~100 mg/ml protein concentration in cytosol, for instance: "Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area." Luby-Phelps, 2000.

2) Results section: Figure 2 clearly shows labeling of the inner leaflet of the PM under normal "untreated" conditions, which disappears upon cholesterol depletion. However, the intense bright puncta observed upon cholesterol depletion may confuse the readers and lead them to think that cholesterol content of an internal organelle like the lysosome increases upon cholesterol depletion! We assume that the authors do not think this is the case. The observed staining is most likely D4 aggregates in cytosol, since the D4 has to go somewhere once membrane cholesterol is depleted. A sentence clarifying this point would be useful.

https://doi.org/10.7554/eLife.38493.007

Author response

Essential revisions:

To make this Comment article as solid as possible, the reviewers request the following essential revisions:

1) The authors show the effect of composition on DAN-D4 binding at a single concentration of D4 (which incidentally is not mentioned in the text). It would be desirable that a binding isotherm be performed/shown that indicates that they are achieving at saturation levels for all compositions tested. It is not clear whether it is the 'availability/accessibility' of cholesterol that is changing or the Kd of D4 for the cholesterol in the membrane. Coupled with the lack of information about the concentrations of the D4 protein, it is hard to come to a firm conclusion.

We now performed DAN-D4 binding curves, particularly for PC with different acyl chain unsaturation (Figure 1B), which is well-documented as the most significant factor influencing cholesterol accessibility. The result is consistent with the conclusion in previous version, and also with existing literature, that D4 accessibility of cholesterol is increased with increasing acyl chain unsaturation. Accessibility will for sure directly affect Kd for cholesterol: the more accessible the cholesterol, the lower the Kd for cholesterol. We apologize for the omission of the details. They are now added in the Materials and methods section.

2) The authors challenge the lack of binding of the DAN- and NR3-labeled D4s to the inner PM leaflet in the Liu et al. study by showing that heterologously expressed mCherry D4s do label the inner leaflet. The impact of this new data piece is modest: transiently transfected fluorescent fusions of D4 probes have been previously reported to localize to the inner plasma membrane leaflet and with the substantial cell-to-cell variation in the labeling pattern observed with transient expression, single cell exemplary images are questionable. The authors suggest that lack of detection of the NR3-D4 at the inner leaflet may be due to low amounts of D4s being microinjected into cells or due to the presence of competing endogenous proteins (the authors use BSA as a case study to make this point). The authors' discussion of this point is not compelling since transient transfection may be even more difficult to control than microinjection in terms of protein dosage and it is not apparent why the transiently expressed protein would behave more correctly. This should be quantitatively addressed by comparing their binding isotherms – it is possible that mCherry D4 variants behave differently from both NR3-D4 variants or the DAN D4 variants.

We agree with the reviewers that the previous explanation we provided as to why the DAN-D4s don’t bind to the PM is rather unsatisfying. In the revised manuscript, we now performed DAN-D4 binding competition with rat liver cytosol (Figure 1C). Result shows that DAN-D4 binding to membrane is highly sensitive to cytosol proteins. Our results suggest that the addition of the lipophilic DAN molecule may increase binding to unknown cytosolic proteins. The mCherry-labeled D4 variants are not likely subjected to such interference. Indeed, a large amount of literate reported that the localization of a different variant, mCherry-D4D434S and its responsiveness to cholesterol extraction, oxidation, supplementation and mislocalization. (see PMID: 25663704, 26572827, 28564600, 28391244 and others for examples).

Nevertheless, in the revised manuscript, we provide additional quantitation and ratiometric analysis for the mCherry-D4 D434A and D434A/A463W in control cells and following 15 min of treatment with 10 mM methyl-b-cyclodextrin. In our experience, using spinning-disc confocal microscopy with live cells we see little heterogeneity with these constructs.

3) The authors need to be cautious in their interpretations regarding point 2 – the exoplasmic leaflet binding may also be subject to interference by proteins.

We appreciate the concern of the reviewers. However, it is worth pointing out that the protein concentration in the cytoplasm is at least 10-fold greater than that in the extracellular medium used for in vitrocell culture experiments. To improve the readability of the manuscript we clarified this point in the text.

4) Liu et al., did test liposomes with substantial amounts of a typical inner leaflet lipid (20 mol% PS, in addition to PC and cholesterol) and also PC/PE/PS/PI containing liposomes, but not systematically with all D4 variants. The authors here are more systematic and additionally show that the acyl chain composition of PC affects D4 binding. They make a valid point but should not mischaracterize Liu et al.

Thank you for the comment, it is well taken, and our language was too strong. In the revised version of the manuscript we have re-written that section. Ultimately, the point that we were trying to make initially remains valid. The binding of D4 to membranes varies significantly, not only between liposomes that mimic the outer vs. inner leaflets, but even more strikingly when acyl chain composition of the liposomes varied. Therefore, it is not appropriate to use these simplified liposomes to as standard curve for interpolation of data obtained from cell-based assays.

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

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Please make the following clarifying revisions.

1) Results section: Please include an additional reference to the large body of earlier work that also suggests ~100 mg/ml protein concentration in cytosol, for instance: "Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area." Luby-Phelps, 2000.

Referenced added.

2) Results section: Figure 2 clearly shows labeling of the inner leaflet of the PM under normal "untreated" conditions, which disappears upon cholesterol depletion. However, the intense bright puncta observed upon cholesterol depletion may confuse the readers and lead them to think that cholesterol content of an internal organelle like the lysosome increases upon cholesterol depletion! We assume that the authors do not think this is the case. The observed staining is most likely D4 aggregates in cytosol, since the D4 has to go somewhere once membrane cholesterol is depleted. A sentence clarifying this point would be useful.

Following sentences are added:

“Noticeably, the liberation of the mCherry-D4 variants from the PM following cholesterol extraction is accompanied by the appearance of bright puncta within the cytosol. This could not be due to a sudden increase in endomembrane cholesterol, as acute cholesterol extraction would only lower cellular cholesterol, including endomembranes. However, without cholesterol-rich membrane to bind, D4 could form aggregates within the cytoplasm or be bound to unidentified membrane structures.”

https://doi.org/10.7554/eLife.38493.008

Article and author information

Author details

  1. Kevin C Courtney

    Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1315-4917
  2. Karen YY Fung

    Keenan Research Centre, St. Michael’s Hospital, Toronto, Canada
    Contribution
    Validation, Investigation, Visualization, Methodology, Performed substantial amount of mCherry experiments, Analyzed data
    Competing interests
    No competing interests declared
  3. Frederick R Maxfield

    Department of Biochemistry, Weill Cornell Medical College, New York, United States
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4396-8866
  4. Gregory D Fairn

    Keenan Research Centre, St. Michael’s Hospital, Toronto, Canada
    Contribution
    Resources, Data curation, Supervision, Investigation, Methodology, Writing—original draft, Writing—review and editing
    For correspondence
    fairng@smh.ca
    Competing interests
    No competing interests declared
  5. Xiaohui Zha

    1. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada
    2. Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Canada
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    xzha@ohri.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2873-3073

Funding

Canadian Institutes of Health Research (Operating Grant MOP-130453)

  • Xiaohui Zha

Natural Sciences and Engineering Research Council of Canada (Discovery Grant RGPIN 40210-2013)

  • Xiaohui Zha

National Institutes of Health (R01 GM123462)

  • Frederick R Maxfield

Canadian Institutes of Health Research (MOP-133656)

  • Gregory D Fairn

The authors declare that there was no funding for this work

Acknowledgements

We would like to acknowledge the valuable insight and feedback that we received from Theodore L. Steck and Yvonne Lange throughout the development of this work. This work was supported by MOP-130453, Canadian Institutes of Health Research (CIHR) and RGPIN 40210–2013, Natural Sciences and Engineering Research Council of Canada (NSERC) (XZ); R01 GM123462 (NIH) (FRM), and MOP-133656, Canadian Institutes of Health Research (CIHR) (GF).

Senior Editor

  1. Philip A Cole, Harvard Medical School, United States

Reviewing Editor

  1. Arun Radhakrishnan, University of Texas Southwestern Medical Center, United States

Publication history

  1. Received: June 4, 2018
  2. Accepted: November 6, 2018
  3. Accepted Manuscript published: November 13, 2018 (version 1)
  4. Version of Record published: November 27, 2018 (version 2)
  5. Version of Record updated: November 29, 2018 (version 3)

Copyright

© 2018, Courtney 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.

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