Phase behavior and domain size in sphingomyelin-containing lipid bilayers

Petruzielo, Robin S.; Heberle, Frederick A.; Drazba, Paul; Feigenson, Gerald W.

doi:10.1016/j.bbamem.2013.01.007

Cited by 117 publications

(172 citation statements)

References 73 publications

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“…Along a tieline the fraction of each coexisting phase is known. Currently, the majority of published phase diagrams describe neutral lipid mixtures (36)(37)(38)(39)48). No published data are available with ternary phase diagrams containing PC/PS/Chol.…”

Section: Resultsmentioning

confidence: 99%

“…One previous study found a nonphysiological enrichment of cholesterol in virions compared to the cellular plasma membrane (33); however, a more thorough study found no significant enrichment in cholesterol (35). Additionally, in model membranes with coexisting raft and non-raft domains at a physiological cholesterol mole fraction of 0.35 to 0.4, cholesterol is present at similar levels in the coexisting domains (36)(37)(38)(39). Also, under some conditions Gag is found in cold detergent-resistant membranes (DRMs) along with raft markers (40)(41)(42)(43).…”

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confidence: 93%

“…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). Each lipid phase has well-defined and uniform properties, such as order and density, throughout the whole domain (25, 51).…”

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confidence: 99%

“…The phase boundaries at the ends of the tieline for composition DSPC/DOPC/Chol (30/45/25) [1,2-distearoyl-sn-glycero-3-phosphocholine-1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol (30/45/ 25)] were determined by fluorescence resonance energy transfer (FRET) as previously described (36,48). FRET samples were prepared at 2% compositional increments except near the phase boundary, where 1% increments were used (37,48). DHE and Fast DiO were chosen as a FRET pair, at 1/100 and 1/700 fractions in total lipid.…”

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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%

mentioning

confidence: 93%

mentioning

confidence: 99%

mentioning

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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“…These constituents are thought to segregate dynamically into nanoscale lateral domains called lipid rafts, which influence vital bioactivity such as signaling events, transport across biological membranes, and regulation of the activity of membrane proteins. 1,2,3,4,5 Due to the complexity of cell membranes, numerous studies have mimicked raft domain properties and phase state using simple model systems comprised of a high melting point (T M ) lipid, a low T M lipid, and sterol. 2,6,7,8 Such ternary mixtures can form coexisting liquid ordered (L o ) / liquid disordered (L d ) phases analogous to liquid-liquid immiscibility in cellular membranes and raft formation.…”

Section: Introductionmentioning

confidence: 99%

Interaction Forces between Lipid Rafts

et al. 2016

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Cellular membranes containing sphingolipids and cholesterol have been shown to self-organize into lipid rafts, specialized domains which host integral membrane proteins and modulate the bioactivity of cells. In this work, force-distance profiles between raft membranes in the liquid ordered phase consisting of singly-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a complex mixture of brain sphingomyelin (BSM), and cholesterol were measured using the surface force apparatus (SFA). Two distinct force profiles were detected corresponding to uniform raft membranes and raft membranes with a higher level of topological membrane defects (heterogeneous) as corroborated by AFM scans. In all cases a weak, longrange electrostatic repulsion was observed with some variation in the surface charge density. The variation in electrostatic repulsion was attributed to charged lipid species primarily from the constituent lipids in the BSM mixture. The adhesion between the uniform raft membranes was comparable to our previous work with pure component, liquid-ordered POPC:DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine):cholesterol membranes. Raft membranes with more topological defects adhered more strongly due to hydrophobic attraction between exposed acyl chains. Even though the rafts were in the liquid-ordered phase and membrane defects were present in the contact region, the raft membranes were stable and no structural rearrangement was observed throughout the measurements. Our findings demonstrate that liquid ordered membranes are stable to mechanical loading and not particularly sensitive to compositional variation.1

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Hydrophobic Interfacing of Fluorescent Membrane Probes

Bayard,

Chen,

García‐Arcos

et al. 2023

ChemistryEurope

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Fluorescent flippers have been introduced as small‐molecule probes to image membrane tension in living systems. While the hydrophilic headgroup region has been modified extensively for intracellular targeting, little is known about the hydrophobic interfacing with the surrounding membrane. To tackle this challenge, the design, synthesis and evaluation of a glutamine‐derived flipper collection is reported. Considering the importance of tension‐induced phase separation for tension imaging, this study is focused on how to modulate the distribution of functional flippers between ordered and disordered microdomains. Also of interest was control over intermembrane transfer without loss of function for the specific labeling of plasma and intracellular membranes. Evidence is presented for a two‐step insertion mechanism through more accessible disordered domains into better matching ordered domains. This process also explains differences between partition coefficients and bioimaging. It is further demonstrated that interdomain and intermembrane distribution can be regulated by hydrophobic interfacing to control brightness in fluorescence lifetime imaging microscopy and responsiveness to membrane tension. Irreversible partitioning inhibits intermembrane transfer and coincides with internalization into cells. These results demonstrate that hydrophobic interfacing can improve probe performance and provide guidelines on how to proceed.

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Phase behavior and domain size in sphingomyelin-containing lipid bilayers

Cited by 117 publications

References 73 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

Interaction Forces between Lipid Rafts

Hydrophobic Interfacing of Fluorescent Membrane Probes

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