Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Comparing physical mechanisms for membrane curvature-driven sorting of BAR-domain proteins.

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.

Unusual Organization of I-BAR Proteins on Tubular and Vesicular Membranes.

Protein-mediated membrane remodeling is a ubiquitous and critical process for proper cellular function. Inverse Bin/Amphiphysin/Rvs (I-BAR) domains drive local membrane deformation as a precursor to large-scale membrane remodeling. We employ a multiscale approach to provide the molecular mechanism of unusual I-BAR domain-driven membrane remodeling at a low protein surface concentration with near-atomistic detail. We generate a bottom-up coarse-grained model that demonstrates similar membrane-bound I-BAR domain aggregation behavior as our recent Mesoscopic Membrane with Explicit Proteins model. Together, these models bridge several length scales and reveal an aggregation behavior of I-BAR domains. We find that at low surface coverage (i.e., low bound protein density), I-BAR domains form transient, tip-to-tip strings on periodic flat membrane sheets. Inside of lipid bilayer tubules, we find linear aggregates parallel to the axis of the tubule. Finally, we find that I-BAR domains form tip-to-tip aggregates around the edges of membrane domes. These results are supported by in vitro experiments showing low curvature bulges surrounded by I-BAR domains on giant unilamellar vesicles. Overall, our models reveal new I-BAR domain aggregation behavior in membrane tubules and on the surface of vesicles at low surface concentration that add insight into how I-BAR domain proteins may contribute to certain aspects of membrane remodeling in cells.

Cell-sized liposomes reveal how actomyosin cortical tension drives shape change.

Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the cell membrane, to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex-membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.

Shape control of lipid bilayer membranes by confined actin bundles.

In living cells, lipid membranes and biopolymers determine each other's conformation in a delicate force balance. Cellular polymers such as actin filaments are strongly confined by the plasma membrane in cell protrusions such as lamellipodia and filopodia. Conversely, protrusion formation is facilitated by actin-driven membrane deformation and these protrusions are maintained by dense actin networks or bundles of actin filaments. Here we investigate the mechanical interplay between actin bundles and lipid bilayer membranes by reconstituting a minimal model system based on cell-sized liposomes with encapsulated actin filaments bundled by fascin. To address the competition between the deformability of the membrane and the enclosed actin bundles, we tune the bundle stiffness (through the fascin-to-actin molar ratio) and the membrane rigidity (through protein decoration). Using confocal microscopy and quantitative image analysis, we show that actin bundles deform the liposomes into a rich set of morphologies. For liposomes having a small membrane bending rigidity, the actin bundles tend to generate finger-like membrane protrusions that resemble cellular filopodia. Stiffer bundles formed at high crosslink density stay straight in the liposome body, whereas softer bundles formed at low crosslink density are bent and kinked. When the membrane has a large bending rigidity, membrane protrusions are suppressed. In this case, membrane enclosure forces the actin bundles to organize into cortical rings, to minimize the energy cost associated with filament bending. Our results highlight the importance of taking into account mechanical interactions between the actin cytoskeleton and the membrane to understand cell shape control.

Actin-membrane linkers: Insights from synthetic reconstituted systems.

At the cell surface, the actin cytoskeleton and the plasma membrane interact reciprocally in a variety of processes related to the remodeling of the cell surface. The actin cytoskeleton has been known to modulate membrane organization and reshape the membrane. To this end, actin-membrane linking molecules play a major role in regulating actin assembly and spatially direct the interaction between the actin cytoskeleton and the membrane. While studies in cells have provided a wealth of knowledge on the molecular composition and interactions of the actin-membrane interface, the complex molecular interactions make it challenging to elucidate the precise actions of the actin-membrane linkers at the interface. Synthetic reconstituted systems, consisting of model membranes and purified proteins, have been a powerful approach to elucidate how actin-membrane linkers direct actin assembly to drive membrane shape changes. In this review, we will focus only on several actin-membrane linkers that have been studied by using reconstitution systems. We will discuss the design principles of these reconstitution systems and how they have contributed to the understanding of the cellular functions of actin-membrane linkers. Finally, we will provide a perspective on future research directions in understanding the intricate actin-membrane interaction.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Gel-assisted formation of giant unilamellar vesicles.

Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.

A biophysical perspective of the regulatory mechanisms of ezrin/radixin/moesin proteins.

Many signal transductions resulting from ligand-receptor interactions occur at the cell surface. These signaling pathways play essential roles in cell polarization, membrane morphogenesis, and the modulation of membrane tension at the cell surface. However, due to the large number of membrane-binding proteins, including actin-membrane linkers, and transmembrane proteins present at the cell surface, the molecular mechanisms underlying the regulation at the cell surface are yet unclear. Here, we describe the molecular functions of one of the key players at the cell surface, ezrin/radixin/moesin (ERM) proteins from a biophysical point of view. We focus our discussion on biophysical properties of ERM proteins revealed by using biophysical tools in live cells and in vitro reconstitution systems. We first describe the structural properties of ERM proteins and then discuss the interactions of ERM proteins with PI(4,5)P2 and the actin cytoskeleton. These properties of ERM proteins revealed by using biophysical approaches have led to a better understanding of their physiological functions in cells and tissues. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00928-0.

Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

PI4P and BLOC-1 remodel endosomal membranes into tubules.

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.

Encapsulation of active cytoskeletal protein networks in cell-sized liposomes.

We demonstrate that cytoskeletal actin-myosin networks can be encapsulated with high efficiency in giant liposomes by hydration of lipids in an agarose hydrogel. The liposomes have cell-sized diameters of 10-20 µm and a uniform actin content. We show by measurements of membrane fluorescence intensity and bending rigidity that the majority of liposomes are unilamellar. We further demonstrate that the actin network can be specifically anchored to the membrane by biotin-streptavidin linkages. These protein-filled liposomes are useful model systems for quantitative studies of the physical mechanisms by which the cytoskeleton actively controls cell shape and mechanics. In a broader context, this new preparation method should be widely applicable to encapsulation of proteins and polymers, for instance, to create polymer-reinforced liposomes for drug delivery.

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

Three-dimensional characterization of active membrane waves on living cells.

We measure the temporal evolution of three-dimensional membrane topography on living fibroblasts and characterize the propagation of membrane waves using a wide-field optical profiling technique. The measured membrane profiles are compared with the numerical results calculated by the active membrane model recently proposed by Shlomovitz and Gov. After the treatments of blebbistatin and latrunculin A separately, the membrane waves disappear and the membrane surfaces are flattened, verifying that the membrane waves are driven by the interactions between myosin II and actin polymerization.

Nanoplasmonic Sensor Detects Preferential Binding of IRSp53 to Negative Membrane Curvature.

Biosensors based on plasmonic nanostructures are widely used in various applications and benefit from numerous operational advantages. One type of application where nanostructured sensors provide unique value in comparison with, for instance, conventional surface plasmon resonance, is investigations of the influence of nanoscale geometry on biomolecular binding events. In this study, we show that plasmonic "nanowells" conformally coated with a continuous lipid bilayer can be used to detect the preferential binding of the insulin receptor tyrosine kinase substrate protein (IRSp53) I-BAR domain to regions of negative surface curvature, i.e., the interior of the nanowells. Two different sensor architectures with and without an additional niobium oxide layer are compared for this purpose. In both cases, curvature preferential binding of IRSp53 (at around 0.025 nm-1 and higher) can be detected qualitatively. The high refractive index niobium oxide influences the near field distribution and makes the signature for bilayer formation less clear, but the contrast for accumulation at regions of negative curvature is slightly higher. This work shows the first example of analyzing preferential binding of an average-sized and biologically important protein to negative membrane curvature in a label-free manner and in real-time, illustrating a unique application for nanoplasmonic sensors.

Membrane curvature induces cardiolipin sorting.

Cardiolipin is a cone-shaped lipid predominantly localized in curved membrane sites of bacteria and in the mitochondrial cristae. This specific localization has been argued to be geometry-driven, since the CL's conical shape relaxes curvature frustration. Although previous evidence suggests a coupling between CL concentration and membrane shape in vivo, no precise experimental data are available for curvature-based CL sorting in vitro. Here, we test this hypothesis in experiments that isolate the effects of membrane curvature in lipid-bilayer nanotubes. CL sorting is observed with increasing tube curvature, reaching a maximum at optimal CL concentrations, a fact compatible with self-associative clustering. Observations are compatible with a model of membrane elasticity including van der Waals entropy, from which a negative intrinsic curvature of -1.1 nm-1 is predicted for CL. The results contribute to understanding the physicochemical interplay between membrane curvature and composition, providing key insights into mitochondrial and bacterial membrane organization and dynamics.

Membrane reshaping by micrometric curvature sensitive septin filaments.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.