Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Protein-membrane interactions: sensing and generating curvature.

Biological membranes are integral cellular structures that can be curved into various geometries. These curved structures are abundant in cells as they are essential for various physiological processes. However, curved membranes are inherently unstable, especially on nanometer length scales. To stabilize curved membranes, cells can utilize proteins that sense and generate membrane curvature. In this review, we summarize recent research that has advanced our understanding of interactions between proteins and curved membrane surfaces, as well as work that has expanded our ability to study curvature sensing and generation. Additionally, we look at specific examples of cellular processes that require membrane curvature, such as neurotransmission, clathrin-mediated endocytosis (CME), and organelle biogenesis.

Tetraspanin proteins in membrane remodeling processes.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Biophysical aspects of migrasome organelle formation and their diverse cellular functions.

The transient cellular organelles known as migrasomes, which form during cell migration along retraction fibers, have emerged as a crutial factor in various fundamental cellular processes and pathologies. These membrane vesicles originate from local membrane swellings, encapsulate specific cytoplasmic content, and are eventually released to the extracellular environment or taken up by recipient cells. Migrasome biogenesis entails a sequential membrane remodeling process involving a complex interplay between various molecular factors such as tetraspanin proteins, and mechanical properties like membrane tension and bending rigidity. In this review, we summarize recent studies exploring the mechanism of migrasome formation. We emphasize how physical forces, together with molecular factors, shape migrasome biogenesis, and detail the involvement of migrasomes in various cellular processes and pathologies. A comprehensive understanding of the exact mechanism underlying migrasome formation and the identification of key molecules involved hold promise for advancing their therapeutic and diagnostic applications.

Membrane remodeling properties of the Parkinson's disease protein LRRK2.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.

Consensus, controversies, and conundrums of P4-ATPases: The emerging face of eukaryotic lipid flippases.

The cryo-EM resolution revolution has heralded a new era in our understanding of eukaryotic lipid flippases with a rapidly growing number of high-resolution structures. Flippases belong to the P4 family of ATPases (type IV P-type ATPases) that largely follow the reaction cycle proposed for the more extensively studied cation-transporting P-type ATPases. However, unlike the canonical P-type ATPases, no flippase cargos are transported in the phosphorylation half-reaction. Instead of being released into the intracellular or extracellular milieu, lipid cargos are transported to their destination at the inner leaflet of the membrane. Recent flippase structures have revealed multiple conformational states during the lipid transport cycle. Nonetheless, critical conformational states capturing the lipid cargo "in transit" are still missing. In this review, we highlight the amazing structural advances of these lipid transporters, discuss various perspectives on catalytic and regulatory mechanisms in the literature, and shed light on future directions in further deciphering the detailed molecular mechanisms of lipid flipping.

Tetraspanner-based nanodomains modulate BAR domain-induced membrane curvature.

The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR domain-induced PM furrows. Here we report a novel function for the Sur7 family of tetraspanner proteins in the regulation of local PM topography. Combining TIRF imaging, STED nanoscopy, freeze-fracture EM and membrane simulations we find that Sur7 tetraspanners form multimeric strands at the edges of PM furrows, where they modulate forces exerted by BAR domain proteins at the furrow base. Loss of Sur7 tetraspanners or Sur7 displacement due to altered PIP2 homeostasis leads to increased PM invagination and a distinct form of membrane tubulation. Physiological defects associated with PM tubulation are rescued by synthetic anchoring of Sur7 to furrows. Our findings suggest a key role for tetraspanner proteins in sculpting local membrane domains. The maintenance of stable PM furrows depends on a balance between negative curvature at the base which is generated by BAR domains and positive curvature at the furrows' edges which is stabilized by strands of Sur7 tetraspanners.

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature.

Multiple membrane-shaping and remodeling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in every cell type. Prominent examples are tetraspanin4 and CD9, which are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here, we revealed that tetraspanin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end, we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodeling processes.

Mechanism of shaping membrane nanostructures of endoplasmic reticulum.

Recent advances in super-resolution microscopy revealed the previously unknown nanoscopic level of organization of endoplasmic reticulum (ER), one of the most vital intracellular organelles. Membrane nanostructures of 10- to 100-nm intrinsic length scales, which include ER tubular matrices, ER sheet nanoholes, internal membranes of ER exit sites (ERES), and ER transport intermediates, were discovered and imaged in considerable detail, but the physical factors determining their unique geometrical features remained unknown. Here, we proposed and computationally substantiated a common concept for mechanisms of all ER nanostructures based on the membrane intrinsic curvature as a primary factor shaping the membrane and ultra-low membrane tensions as modulators of the membrane configurations. We computationally revealed a common structural motif underlying most of the nanostructures. We predicted the existence of a discrete series of equilibrium configurations of ER tubular matrices and recovered the one corresponding to the observations and favored by ultra-low tensions. We modeled the nanohole formation as resulting from a spontaneous collapse of elements of the ER tubular network adjacent to the ER sheet edge and calculated the nanohole dimensions. We proposed the ERES membrane to have a shape of a super flexible membrane bead chain, which acquires random walk configurations unless an ultra-low tension converts it into a straight conformation of a transport intermediate. The adequacy of the proposed concept is supported by a close qualitative and quantitative similarity between the predicted and observed configurations of all four ER nanostructures.

Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Cholesterol and M2 Rendezvous in Budding and Scission of Influenza A Virus.

The cholesterol of the host cell plasma membrane and viral M2 protein plays a crucial role in multiple stages of infection and replication of the influenza A virus. Cholesterol is required for the formation of heterogeneous membrane microdomains (or rafts) in the budozone of the host cell that serves as assembly sites for the viral components. The raft microstructures act as scaffolds for several proteins. Cholesterol may further contribute to the mechanical forces necessary for membrane scission in the last stage of budding and help to maintain the stability of the virus envelope. The M2 protein has been shown to cause membrane scission in model systems by promoting the formation of curved lipid bilayer structures that, in turn, can lead to membrane vesicles budding off or scission intermediates. Membrane remodeling by M2 is intimately linked with cholesterol as it affects local lipid composition, fluidity, and stability of the membrane. Thus, both cholesterol and M2 protein contribute to the efficient and proper release of newly formed influenza viruses from the virus-infected cells.

Dendrite tapering actuates a self-organizing signaling circuit for stochastic filopodia initiation in neurons.

How signaling units spontaneously arise from a noisy cellular background is not well understood. Here, we show that stochastic membrane deformations can nucleate exploratory dendritic filopodia, dynamic actin-rich structures used by neurons to sample its surroundings for compatible transcellular contacts. A theoretical analysis demonstrates that corecruitment of positive and negative curvature-sensitive proteins to deformed membranes minimizes the free energy of the system, allowing the formation of long-lived curved membrane sections from stochastic membrane fluctuations. Quantitative experiments show that once recruited, curvature-sensitive proteins form a signaling circuit composed of interlinked positive and negative actin-regulatory feedback loops. As the positive but not the negative feedback loop can sense the dendrite diameter, this self-organizing circuit determines filopodia initiation frequency along tapering dendrites. Together, our findings identify a receptor-independent signaling circuit that employs random membrane deformations to simultaneously elicit and limit formation of exploratory filopodia to distal dendritic sites of developing neurons.

Membrane bending by protein phase separation.

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region.

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms.

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties.

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.

Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors.

Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some curvature-sensing molecules not only detect membrane deformation but can also alter the amplitude of forces that caused to shape changes in the first place. This dual ability of sensing and inducing membrane deformation leads to the formation of curvature-dependent self-organizing signaling circuits. How these cell-autonomous circuits are affected by auxiliary parameters from inside and outside of the cell has remained largely elusive. Here, we explore how such factors modulate self-organization at the micro-scale and its emerging properties at the macroscale.

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.