Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast
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Abstract
Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.
Summary statement
Yeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.
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###Reviewer #3:
The manuscript by Mioka et al. is the synthesis of a lot of well executed experiments examining a "void" zone in the plasma membrane of yeast cells lacking phosphatidylserine. The authors demonstrate that this is a specialized micron-size domain with many intriguing properties. However, there are several issues that limit my enthusiasm. Some of the experiments are misinterpreted, and there are also inconsistencies and inaccuracies in the text. In my opinion Figure 6 and Figure7 provide little benefit from the primary findings of the paper.
Other concerns:
The void zones shown are more prevalent at 37C than 30C. This is opposite to the other micron sized phase separation in the yeast vacuole (Rayermann et al., 2017). If this is a Lo domain then rapid oscillations in temperature should control the reversible assembly and …
###Reviewer #3:
The manuscript by Mioka et al. is the synthesis of a lot of well executed experiments examining a "void" zone in the plasma membrane of yeast cells lacking phosphatidylserine. The authors demonstrate that this is a specialized micron-size domain with many intriguing properties. However, there are several issues that limit my enthusiasm. Some of the experiments are misinterpreted, and there are also inconsistencies and inaccuracies in the text. In my opinion Figure 6 and Figure7 provide little benefit from the primary findings of the paper.
Other concerns:
The void zones shown are more prevalent at 37C than 30C. This is opposite to the other micron sized phase separation in the yeast vacuole (Rayermann et al., 2017). If this is a Lo domain then rapid oscillations in temperature should control the reversible assembly and disassembly. This should be examined.
It's odd to me that the filipin signal has "thickness" beyond what you would expect if it was confined to a bilayer. In other experiments it appears that the cytosolic fluorescence is also quenched in the vicinity of the voids. This is problematic as every GFP construct examined on the cytosolic side of the PM is excluded. Perhaps these cells actually have ergosterol crystals (a 3D structure) rather than a Lo domain within the bilayer. Given the importance of cholesterol crystals in being a "danger" signal and activating inflammasomes it could be worth examining. This would require specialized imaging techniques.
Spira et al., (2012, NCB). Highlighted the patchwork nature of the plasma membrane. With Pma1 and Ras2 being excluded from one another and proteins with similar TMDs tend to colocalize. This article should be included in the discussion to help place these findings in a greater context. Yet here all of the constructs that are examined are excluded from the void zones. This again suggests to me that this is different from an Lo domain. In the cho1 cells that do not have obvious voids, what is the localization and overlap a few of the well characterized markers Ras2, Pma1, Sur7, Bio5?
Figure 1B shows 40% of cells grown overnight at 37C have voids but Figure 2C shows that they are lost after ~15h. This seems inconsistent.
The authors state that psd1 psd2 are PE-deficient and cho2 opi3 are PC-deficient in the figure. This is incorrect.
Figure 3C is not convincing. Images on the right have substantially more red pixels and so positions where there were voids at 0 min now have a bit of green at 25 min. I also don't understand how the ergosterol rich region is able to quench signal in the cytosol. Is this an extended focus representation of multiple slices?
GPI-linked proteins are crosslinked to the cell wall. The authors' conclusions cannot be drawn from this experiment. The authors could potentially do the same experiment in spheroplasts.
Alternatively, adding rhodamine-PE to the cells could be used to assess the partitioning in the outer leaflet.
The significance of the vacuole - void contact is unclear. Typically, ~50% of the PM is in close apposition to cER in yeast. In mammalian cells it is known that cortical actin can restrict ER-PM contact sites formation. Thus, it could simply be that in the absence of cER that the Vacuole will come in close proximity to the PM. This can be tested by using a strain deficient in reticulons or the so-called delta tether or delta super-tether cells. If these cells also display Vac - PM contacts, then I don't see the relevance of including this figure in this study.
Vacuole - void contacts are seen in roughly 50% of the cells with voids. In the cells that don't have this V-V contact do they have the nucleus or nER in contact with the PM? This is related to the above point. Is this simply a result of removing the cER and making the PM available?
Figure 7 is unnecessary and just makes things more complicated. It actually detracts from the main findings since it is just a collection of observations. For instance, how would loss of the HOPS complex prevent Lo phase separation in the plasma membrane? Do these cells have less total cellular or plasmalemmal ergosterol? Do the levels of complex sphingolipids change?
Provide a reference or a direct measurement showing that growing cells in pH7.0 medium impacts the cytosolic pH.
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###Reviewer #2:
This study shows that plasma membrane (PM) voids, regions devoid of proteins, form in cells lacking phosphatidylserine (PS). It argues these regions are enriched in ergosterol and are liquid ordered. Domain formation is reversible and may require ergosterol and sphingolipids for formation. A number of genes that disrupt void formation are also identified. The study proposes that PS prevents the formation of void zones by interacting with ergosterol. Overall, the study is well done and makes a persuasive case that that protein-free voids form in the PM and do not seem to affect cell growth; a fascinating discovery. There are, however, two weaknesses in the study that reduce its impact. One is that it does not show PS is directly involved in void formation or that void zone formation is driven by PS-ergosterol …
###Reviewer #2:
This study shows that plasma membrane (PM) voids, regions devoid of proteins, form in cells lacking phosphatidylserine (PS). It argues these regions are enriched in ergosterol and are liquid ordered. Domain formation is reversible and may require ergosterol and sphingolipids for formation. A number of genes that disrupt void formation are also identified. The study proposes that PS prevents the formation of void zones by interacting with ergosterol. Overall, the study is well done and makes a persuasive case that that protein-free voids form in the PM and do not seem to affect cell growth; a fascinating discovery. There are, however, two weaknesses in the study that reduce its impact. One is that it does not show PS is directly involved in void formation or that void zone formation is driven by PS-ergosterol interactions, as stated in the abstract and elsewhere. This could be addressed in vitro using GUVs or supported bilayers. I realize these experiments are challenging, but they could add significant mechanistic insight. The second major weakness of the study is that it does not demonstrate PM void zones occur in wild-type cells in response to stress or in some growth conditions. There are other, more minor concerns.
- There is no direct demonstration that the void domains are ordered. This could be shown using order sensitive dyes like Laurdan. Further evidence could be provided by directly measuring diffusion rates of fluorescent lipids in the void zones compared to the rest of the PM. In addition, if the void domains are ordered, it should be possible to show they melt and reform as cells are heated and cooled.
2)The role of Osh6 and Osh 7 in void formation should be assessed since these proteins are thought to be necessary to maintain PS enrichment in the PM, at least in some growth conditions.
The investigation of void zone-vacuoles (V-V) contact sites is not well explained. It is not clear what is being proposed. How would contact sites promote void zone formation? Are they sites of lipid transfer and, if so, how would that affect void-zone formation? Or is some other mechanism being proposed?
It is not clear what the mutant analysis adds to the story. Do the mutations affect PS levels in the PM? If that is what is being proposed it should be tested. Or do the authors think the mutants affect void zone formation by some other mechanism?
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###Reviewer #1:
The manuscript by Mioka et al. presents an interesting and puzzling observation. The authors showed the existence of a so-called "void zone" in PS-deficient cho1∆ cells. This void zone is a membrane region devoid of proteins and with a specific lipid composition, which the authors suggest to be a microscopic liquid-ordered domain. They also tested different stress conditions and found some that prevented void zone formation in cho1∆ cells. The authors propose that PS is a key lipid in preventing macroscopic raft-like domain formation in WT cells. Although it is unclear whether such PM void zones can appear in WT cells under any stress conditions (hence a caution note on the physiological relevance of the findings herein presented), the authors' proposal that PS in WT cells can suppress the formation of macroscopic lipid …
###Reviewer #1:
The manuscript by Mioka et al. presents an interesting and puzzling observation. The authors showed the existence of a so-called "void zone" in PS-deficient cho1∆ cells. This void zone is a membrane region devoid of proteins and with a specific lipid composition, which the authors suggest to be a microscopic liquid-ordered domain. They also tested different stress conditions and found some that prevented void zone formation in cho1∆ cells. The authors propose that PS is a key lipid in preventing macroscopic raft-like domain formation in WT cells. Although it is unclear whether such PM void zones can appear in WT cells under any stress conditions (hence a caution note on the physiological relevance of the findings herein presented), the authors' proposal that PS in WT cells can suppress the formation of macroscopic lipid domains is an interesting hypothesis that deserves to be followed to my opinion. Finally, the authors start a search for genes required for void zone formation, which is interesting in my opinion, and although only partial conclusions from that can be drawn at the moment, I think this a promising way to study the mechanisms and maybe physiological relevance of void zone formation in the future.
I have some concerns, especially on the fact that they seem to claim that the void zone is a liquid-ordered domain (if so, it should look more circular and not as they show they look like).
Major concerns:
The authors say that Lo domains are completely depleted of transmembrane (TM) proteins. However, there are many reports (e.g. from the Levental lab), where TM proteins with "raft" affinity have been shown. The authors should express some of these raft TM markers and check whether they partition or not into the void zone.
The claim that the void zone is a liquid-ordered (Lo) domain, I do not think there is enough experimental evidence for that. In particular:
-Line 82: the fact that the domains are not circular isn't this against a Lo phase and favor a more gel/solid phase? Have the authors seen fusion of void zone domains in live cells?
-Line 84: does FM4 partition equally to Lo and Ld (liquid-disordered) domains in vitro? What about gel-like domains?
-Lines 304-307: along the same lines, this is true for some proteins, although there are TM proteins that have been shown to be targeted specifically to Lo regions in GMPVs.
-The fact that the void zone appears at high temperature is puzzling if compared to standard liquid-ordered domains.
-Line 687: these observations are also compatible with gel-like domains.
-Is it possible to do some dynamic measurements of dye diffusion in void zones? FRAP? Single particle tracking?
- Many trafficking routes/genes are required for void zone formation. What about for the stability/maintenance? Could the authors provide dynamic anchor-away or degron-tagging of some of these candidates to test whether void zones disappear upon depletion of these proteins?
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
This manuscript shows the interesting observation that plasma membranes in yeast cells lacking phosphatidylserine (PS) present differentiated regions, the so-called "void zones". Void zones are devoid of proteins and have a specific lipid composition (are enriched in ergosterol), which the authors suggest to be a microscopic liquid-ordered domain. Void zone formation is reversible and may require ergosterol and sphingolipids for its formation. They also tested different stress conditions and found some that …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
This manuscript shows the interesting observation that plasma membranes in yeast cells lacking phosphatidylserine (PS) present differentiated regions, the so-called "void zones". Void zones are devoid of proteins and have a specific lipid composition (are enriched in ergosterol), which the authors suggest to be a microscopic liquid-ordered domain. Void zone formation is reversible and may require ergosterol and sphingolipids for its formation. They also tested different stress conditions and found some that prevented void zone formation in cho1∆ cells. The authors propose that PS is a key lipid in preventing macroscopic raft-like domain formation in WT cells, in particular by interacting with ergosterol. Finally, a study for genes that disrupt void formation is also presented.
As you will see all the reviewers acknowledge that the manuscript presents high quality experiments and potentially very interesting discoveries. However, they all coincide in that the story has some weaknesses.
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