Phase Separation in Lipid Membranes

Heberle, Frederick A.; Feigenson, Gerald W.

doi:10.1101/cshperspect.a004630

Cited by 215 publications

(188 citation statements)

References 57 publications

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“…On any point of total sample composition within the LdϩLo coexistence region, the tieline through this point (dashed line in Fig. 6B) intercepts the phase boundaries, connecting the compositions of the two coexisting phases that are in equilibrium (46). Along a tieline the fraction of each coexisting phase is known.…”

Section: Resultsmentioning

confidence: 99%

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Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Wen

Dick

Feigenson

et al. 2016

J Virol

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The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCERetroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.T he retroviral structural protein Gag provides the primary driving force for virus assembly at the inner leaflet of the plasma membrane (PM), and Gag alone is sufficient to assemble into virus-like particles (VLPs) in cells (1) and also in vitro (2). Gag is synthesized in the cytosol and then is targeted to the PM by the N-terminal domain, MA. How Gag interacts with the inner leaflet of the PM and how the biophysical properties of the PM...

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Section: Resultsmentioning

confidence: 99%

“…In vitro, liquid-liquid phase separation can be observed with lipid-only mixtures of as few as three components (45,46). Understanding biological membrane phases is facilitated by lipid compositional phase diagrams (36,37,(47)(48)(49)(50).…”

Section: Importancementioning

confidence: 99%

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Wen

Dick

Feigenson

et al. 2016

J Virol

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“…Similar mixtures are known to give rise to large-scale phase separation consisting of ordered domains (enriched in saturated lipids) in a liquid disordered matrix (enriched in unsaturated lipids) (42). In particular, we prepared (i) a "DSPS sample" (DSPS/DSPC/ DOPC/cholesterol at a 15:30:45:10 molar ratio), in which PS was concentrated in small ordered domains, and (ii) a "DOPS sample" (DOPS/DSPC/DOPC/cholesterol at a 15:30: 45:10 molar ratio), in which PS was distributed all over the liquid disordered matrix.…”

Section: Resultsmentioning

confidence: 99%

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

Bobone

Hilsch

Storm

et al. 2017

J Virol

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Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.KEYWORDS influenza, assembly, confocal microscopy, fluorescence image analysis, lipid rafts, matrix protein, model membranes, phosphatidylserine, plasma membrane I nfluenza A virus (IAV) is a major burden to human health and remains a prominent cause of mortality worldwide (1, 2). Despite significant research efforts, detailed knowledge about the molecular mechanisms involved in IAV infection is still limited. A deeper understanding of the virus machinery and its interaction with the host is

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“…26 Protein constructs were subcloned into the pN1 vector with a strong CMV IE promoter. Fulllength and truncated C-Src-GFP fusions (C-Src 16 -GFP and C-SrcFL-GFP) were engineered from the mouse C-Src gene from Addgene (Plasmid #13663). CD52-GFP genes were gifts from Dr. Björn Lillemeier and Dr. Mark Davis (Stanford University).…”

Section: Cloning and Cell Culture/transfection Of Anchored Gfp Fusionmentioning

confidence: 99%

“…The degree to which lipid miscibility phase behavior contributes to the structure of the complete live cell membrane remains controversial. [12][13][14][15][16][17][18] In this report, we measure the collective mobility of a number of membrane components as a function of temperature in living cell membranes by fluorescence correlation spectroscopy (FCS). [19][20] Since collective mobility exhibits a discontinuity as a system passes through a miscibility phase transition, [21][22] these observations are expected to reveal the phase transition, even in cases where the phase domains are too small to resolve by direct imaging.…”

Section: Introductionmentioning

confidence: 99%

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

et al. 2015

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Lipid:cholesterol mixtures derived from cell membranes, as well as their synthetic reconstitutions, exhibit well defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid:cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid phase separation is not generally observed in cell membranes and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte-Carlo and atomistic simulations on model lipid membrane systems. However, temperature dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes show a consistently smooth increase of the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14 -37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical paramters, such as tension, in the membrane.

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Phase Separation in Lipid Membranes

Cited by 215 publications

References 57 publications

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

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