Giant Plasma Membrane Vesicles Research Articles

Whole-GUV patch-clamping

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains

Current models of lipid rafts propose that lipid domains exist as nanoscale compositional fluctuations and these fluctuations can potentially be stabilized into larger domains, consequently better compartmentalizing cellular functions. However, the mechanisms governing stabilized raft assembly and function remain unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) that binds up to five GM1 glycosphingolipids to enter host cells. We show that when applied to cell-derived giant plasma membrane vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning to the ordered phase compared to wild-type CTxB with five binding sites. Moreover, monovalent CTxB does not stabilize membrane domains, unlike wild-type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a lipid crosslinking mechanism and establish a role for crosslinking in the partitioning of CTxB to ordered domains.

Diffusion of Single-Pass Transmembrane Receptors: From the Plasma Membrane into Giant Liposomes

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4–5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

Selective Targeting and Restrictive Damage for Nonspecific Cells by Pulsed Laser-Activated Hyaluronan-Gold Nanoparticles.

Herein, we describe an approach that immobilizes low-molecular-weight hyaluronic acid (low-MW HA) on the surface of gold nanoparticles (GNPs), which can serve as a cellular probe and photodamage media, to evaluate the selectivity and efficiency of HA-based GNPs (HGNPs) as a mediator of laser-induced photothermal cell damage. In addition, it is known that solid tumors contain a higher content of low-MW HA than normal tissues. Thus, we used low-MW HA rather than high-MW HA used in other studies. In the present study, we conjugated low-MW HA, which is a linear polysaccharide with a disaccharide repeat unit, to prevent a reduction of the ligand-receptor binding efficiency in contrast to the conjugation of protein or peptides, which have unique three-dimensional structures. Three cell lines-MDA-MB-435 S (with CD44), MDA-MB-453 and NIH/3T3 (both are without CD44)-were investigated in the study, and qualitative observations were conducted by dark-field microscopy and laser scanning confocal microscopy (LSCM). In addition, quantitative measurements calculated using inductively coupled plasma emissions were taken for comparison. Our results showed that within the same treatment time, the uptake dosage of HGNPs by the MDA-MB-435 S cells was higher than that by the MDA-MB-453 and NIH 3T3 cells. Meanwhile, HGNPs uptake by the untreated MDA-MB-435 S cells was higher than that of MDA-MB-435 S cells with CD44 blocked by antibodies or silencing CD44 expression. This result implies that receptor-mediated endocytosis can enhance the cellular uptake of HGNPs. In addition, when exposed to a low-power pulsed laser, the former cell morphologies showed a more laser-induced giant plasma membrane vesicles (GPMV) than the latter morphologies. Therefore, this study utilized the specific photothermal property of HA-modified GNPs with laser-induced blebs to create a possible new method for medical applications.

Motility and stem cell properties induced by the epithelial-mesenchymal transition require destabilization of lipid rafts.

The Epithelial-Mesenchymal Transition (EMT) is a developmental program that provides cancer cells with the characteristics necessary for metastasis, including increased motility and stem cell properties. The cellular and molecular mechanisms underlying this process are not yet fully understood, hampering efforts to develop therapeutics. In recent years, it has become apparent that EMT is accompanied by wholesale changes in diverse signaling pathways that are initiated by proteins at the plasma membrane (PM). The PM contains thousands of lipid and protein species that are dynamically and spatially organized into lateral membrane domains, an example of which are lipid rafts. Since one of the major functions of rafts is modulation of signaling originating at the PM, we hypothesized that the signaling changes occurring during an EMT are associated with alterations in PM organization. To test this hypothesis, we used Giant Plasma Membrane Vesicles (GPMVs) to study the organization of intact plasma membranes isolated from live cells. We observed that induction of EMT significantly destabilized lipid raft domains. Further, this reduction in stability was crucial for the maintenance of the stem cell phenotype and EMT-induced remodeling of PM-orchestrated pathways. Exogenously increasing raft stability by feeding cells with ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) repressed these phenotypes without altering EMT markers, and inhibited the metastatic capacity of breast cancer cells. Hence, modulating raft properties regulates cell phenotype, suggesting a novel approach for targeting the impact of EMT in cancer.

Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes

Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways — endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

Using Giant Plasma Membrane Vesicles from Cells to Form Supported Lipid Bilayers

Previous studies have established chemical vesiculation and osmotic stress methods to obtain giant plasma membrane vesicles (GPMVs) from cells. The GPMVs are the blebs directly from the cell plasma membrane and contain most of the biological components in the plasma membrane including the lipids and the embedded membrane proteins. Being able to use GPMVs to form supported lipid bilayers can allow numerous biosensing applications. However, GPMVs do not easily rupture on conventional supports to form supported lipid bilayers because of their high protein and cholesterol contents. Here, we demonstrate the possibility to use air-water interface to break GPMVs to form supported lipid bilayers. We constructed micro-grating structured features on the support to control the air-water interface in a way so that the interface can rupture the GPMVs but does not peel off the deposited GPMV patches. The method provides us a way to form supported lipid bilayers with native plasma membrane proteins. Incorporating the built platforms with some surface analytical tools could allow us to study membrane proteins in their native environment.

Structural Determinants of Raft Partitioning for Single-Pass Transmembrane Proteins

Transmembrane proteins (TMPs) comprise ∼30% of the mammalian proteome and mediate nearly all functions of the cell. The subcellular localization, trafficking, signalling, and enzymatic activity of many TMPs is regulated by their association with lipid-driven lateral membrane domains known as lipid rafts. However, the structural determinants of TMP partitioning to raft domains remain almost unknown. We hypothesized that structural features of the transmembrane domain (TMD) of single-pass TMPs would guide raft partitioning and raft-dependent sub-cellular trafficking. To explore TMD-dependent raft partitioning, we isolated plasma membranes as Giant Plasma Membrane Vesicles (GPMVs), a model system that allows direct observation of protein partitioning between raft/non-raft phases by fluorescence microscopy. We generated 96 different fluorescent mutants of a model TMP and quantified their partitioning as a function of TMD features, namely hydrophobic length and accessible surface area. Raft partitioning was related to TMD length, with longer TMDs imparting greater raft association, confirming previously untested predictions. Investigation of 74 mutants with constant TMD length revealed that raft partitioning was also inversely related to the TMD accessible surface area, with larger surface areas preferring the non-raft phase. To explain these results, we present a simple physical model wherein raft partitioning is driven by differences in interfacial energy between the TMD and its surrounding lipid matrix in raft and non-raft phases. This model is in quantitative agreement with experimental observations and provides first predictions of protein-lipid surface tensions differences between coexisting membrane phases. The model could successfully connect predicted raft partitioning with subcellular localization of single-pass TMPs in Eukaryota. These results point the way to a general rule for raft partitioning of TMPs and underline the central role of membrane domains in cellular traffic.

The Biophysics of Living Membranes: Protein Partitioning and Functional Differentiation in Ordered Plasma Membrane Domains

The lipid raft hypothesis posits that ordered, lateral membrane domains are important for the regulation of plasma membrane (PM) structure and function in eukaryotes. Giant Plasma Membrane Vesicles (GPMVs) are isolated plasma membranes that microscopically separate into coexisting ordered and disordered phases, facilitating experimental analysis of the composition and physical properties of ordered raft domains in biological membranes. In these studies, we analyze the biophysical and lipidomic differentiation of the plasma membrane in Mesenchymal Stem Cells (MSC) as the cells undergo differentiation into adipocytes and osteoblasts. During differentiation, dramatic remodeling the PM lipidome, including lipid acyl chain length and unsaturation, results inchanges to membrane order and phase separation. These observations elucidate the compositional determinants of biophysical properties in biological membranes, as well as identify lineage-specific PM features. These results facilitate rational remodeling of membrane phenotypes to direct differentiation, as supplementation with ꙍ-3 docosahexaneoic acid (DHA) promoted the osteoblastic PM phenotype and potentiated osteogenic differentiation. The differences in the physical properties of the coexisting domains lead to preferential protein partitioning between them. We evaluate the structural determinants of raft partitioning of a model transmembrane protein, and find that raft phase partitioning is related to features of the protein's transmembrane domain (TMD), namely the hydrophobic length and surface area of the TMD. Longer TMDs impart greater raft association while TMDs with larger, bulkier amino acid side chains prefer the non-raft phase. We present a simple physical model wherein raft partitioning is driven by phase-dependent differences in interfacial energy between the TMD and its surrounding lipid matrix, and find excellent quantitative agreement with observations that provide the first predictions of protein-lipid surface tension. These results point the way to a general rule for raft partitioning of transmembrane proteins.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.

Clusters of Cholera Toxin B Subunit on the Outer Leaflet Stabilize Lipid Heterogeneity on the Inner Leaflet of B Cell Membranes

Protein sorting based on liquid-ordered or liquid-disordered phase preference is readily observed in giant plasma membrane vesicles (GPMVs) at low temperatures but is not observed directly in the intact cells from which they are derived. Here we utilize two-color super-resolution microscopy to quantify the sorting of two minimal inner leaflet anchored-peptides proximal to clusters of cholera toxin B subunit (CTxB) in chemically fixed CH27 B cells. One peptide, called PM, is anchored through saturated palmitoyl and myristoyl acylations, giving it a strong preference for more ordered lipids, while the second peptide, called GG, is anchored through an unsaturated and branched geranylgeranyl modification that gives it a strong preference for more disordered lipids. We find that the local density of PM is increased in the vicinity of CTxB clusters while the local density of GG is reduced in the vicinity of CTxB clusters when compared to the average density of peptides on the cell surface. These results indicate that clustering of CTxB on the outer leaflet affects the lateral organization of proteins anchored to the inner leaflet in a way that depends on the anchoring structure. Ongoing work is aimed at probing the membrane's ability to sort proteins based on the phase preference of their membrane anchor in response to biochemical perturbations and changes in temperature, with the goal of distinguishing possible physical bases of this lipid-mediated membrane heterogeneity.

Specific Adhesion of Giant Plasma Membrane Vesicles to Surface-Immobilized SIRPα by Membrane Reconstituted “Marker of Self” Signaling Protein CD47

Model membranes consisting of pure lipid components have clearly advanced the understanding of biological cell membranes. However, the reconstitution of membrane proteins into model lipid bilayers is not straightforward and requires specific strategies for each protein system. In contrast, membranes of living cells have a complex composition with a multitude of different lipid components, proteins and sugars. In this work, we show that membrane blebs derived from the plasma membrane of living cells, also called giant plasma membrane vesicles, can be used to study the interaction of the membrane proteins CD47 and SIRPα, which are involved in immune signaling. Membrane blebs are an interesting model because they exhibit the compositional heterogeneity of the plasma membrane but their morphological behavior can still be described by a few parameters such as bending rigidity, reduced volume and tension. Specifically, we study the interaction of CD47 reconstituted in blebs and surface-immobilized SIRPα. Using this model system we explore membrane-mediated cooperative binding effects under different biologically relevant conditions. We find that acidic environment decreases the binding affinity of membrane-reconstituted CD47 to SIRPα. Finally, we discuss implications for in-vitro cancer models. Financial support from the Deutsche Forschungsgemeinschaft (DFG) via the IRTG 1524 is gratefully acknowledged.

Investigating Large Scale Liquid-Liquid Phase Separation in a Biological Membrane

Toulmay and Prinz recently imaged micron-scale inhomogeneity of labeled proteins and labeled lipids within membranes of vacuole organelles of yeast cells (Toulmay & Prinz, J. Cell Biol., 2013). Such inhomogeneities may provide a mechanism for the protein organization on cell membranes necessary for signal transduction and viral pathogenesis. An outstanding question is whether this inhomogeneity is due to demixing of liquid-ordered (Lo) and liquid-disordered (Ld) membrane phases. Such phases have been previously well characterized in both model vesicles composed of at least three lipid types as well as in Giant Plasma Membrane Vesicles derived from cells. Here we apply techniques of physical chemistry (through changes in temperature and osmotic pressure) to intact yeast vacuole membranes. We use fluorescence microscopy to assess if the vacuole membranes react in a manner consistent with miscibility phase behavior, which would provide the first proof of liquid-liquid phase separation in an unperturbed cell membrane.

Stabilization of membrane necks by adhesive particles, substrate surfaces, and constriction forces.

Membrane remodelling processes involving the formation and fission of small buds require the formation and closure of narrow membrane necks, both for biological membranes and for model membranes such as lipid bilayers. The conditions required for the stability of such necks are well understood in the context of budding of vesicles with bilayer asymmetry and/or intramembrane domains. In many cases, however, the necks form in the presence of an adhesive surface, such as a solid particle or substrate, or the cellular cortex itself. Examples of such processes in biological cells include endocytosis, exocytosis and phagocytosis of solid particles, the formation of extracellular and outer membrane vesicles by eukaryotic and prokaryotic cells, as well as the closure of the cleavage furrow in cytokinesis. Here, we study the interplay of curvature elasticity, membrane-substrate adhesion, and constriction forces to obtain generalized stability conditions for closed necks which we validate by numerical energy minimization. We then explore the consequences of these stability conditions in several experimentally accessible systems such as particle-filled membrane tubes, supported lipid bilayers, giant plasma membrane vesicles, bacterial outer membrane vesicles, and contractile rings around necks. At the end, we introduce an intrinsic engulfment force that directly describes the interplay between curvature elasticity and membrane-substrate adhesion.

Membrane Transition Temperature Determines Cisplatin Response.

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.

Cell membrane softening in human breast and cervical cancer cells

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

Pharmacological targeting of membrane rigidity: implications on cancer cell migration and invasion

The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes.

Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine-containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol-rich domains in a GPMV model, thus documenting the potential of our approach for membrane-partitioning studies.

Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

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 shows a consistently smooth increase in 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 parameters, such as tension, in the membrane.

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.