Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Structural analysis of the P132L disease mutation in caveolin-1 reveals its role in the assembly of oligomeric complexes.

Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a disease-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with WT CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.

Lipid Peroxidation Drives Liquid-Liquid Phase Separation and Disrupts Raft Protein Partitioning in Biological Membranes.

The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure and physicochemical properties of lipids, leading to bilayer thinning, altered fluidity, and increased permeability of membranes in model systems. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances the phase separation propensity of GPMVs into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains and increases the relative abundance of the disordered phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both Lo and Ld domains, and translocation of multiple classes of raft proteins out of ordered domains. These findings indicate that the peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease and thus serve as potential targets for therapeutic intervention.

What's past is prologue: FRAP keeps delivering 50 years later.

Fluorescence recovery after photobleaching (FRAP) has emerged as one of the most widely utilized techniques to quantify binding and diffusion kinetics of biomolecules in biophysics. Since its inception in the mid-1970s, FRAP has been used to address an enormous array of questions including the characteristic features of lipid rafts, how cells regulate the viscosity of their cytoplasm, and the dynamics of biomolecules inside condensates formed by liquid-liquid phase separation. In this perspective, I briefly summarize the history of the field and discuss why FRAP has proven to be so incredibly versatile and popular. Next, I provide an overview of the extensive body of knowledge that has emerged on best practices for quantitative FRAP data analysis, followed by some recent examples of biological lessons learned using this powerful approach. Finally, I touch on new directions and opportunities for biophysicists to contribute to the continued development of this still-relevant research tool.

Template-free prediction of a new monotopic membrane protein fold and assembly by AlphaFold2.

AlphaFold2 (AF2) has revolutionized the field of protein structural prediction. Here, we test its ability to predict the tertiary and quaternary structure of a previously undescribed scaffold with new folds and unusual architecture, the monotopic membrane protein caveolin-1 (CAV1). CAV1 assembles into a disc-shaped oligomer composed of 11 symmetrically arranged protomers, each assuming an identical new fold, and contains the largest parallel ß-barrel known to exist in nature. Remarkably, AF2 predicts both the fold of the protomers and the interfaces between them. It also assembles between seven and 15 copies of CAV1 into disc-shaped complexes. However, the predicted multimers are energetically strained, especially the parallel ß-barrel. These findings highlight the ability of AF2 to correctly predict new protein folds and oligomeric assemblies at a granular level while missing some elements of higher-order complexes, thus positing a new direction for the continued development of deep-learning protein structure prediction approaches.

The building blocks of caveolae revealed: caveolins finally take center stage.

The ability of cells to divide, migrate, relay signals, sense mechanical stimuli, and respond to stress all rely on nanoscale invaginations of the plasma membrane known as caveolae. The caveolins, a family of monotopic membrane proteins, form the inner layer of the caveolar coat. Caveolins have long been implicated in the generation of membrane curvature, in addition to serving as scaffolds for signaling proteins. Until recently, however, the molecular architecture of caveolins was unknown, making it impossible to understand how they operate at a mechanistic level. Over the past year, two independent lines of evidence - experimental and computational - have now converged to provide the first-ever glimpse into the structure of the oligomeric caveolin complexes that function as the building blocks of caveolae. Here, we summarize how these discoveries are transforming our understanding of this long-enigmatic protein family and their role in caveolae assembly and function. We present new models inspired by the structure for how caveolins oligomerize, remodel membranes, interact with their binding partners, and reorganize when mutated. Finally, we discuss emerging insights into structural differences among caveolin family members that enable them to support the proper functions of diverse tissues and organisms.

Peripheral myelin protein 22 preferentially partitions into ordered phase membrane domains.

The ordered environment of cholesterol-rich membrane nanodomains is thought to exclude many transmembrane (TM) proteins. Nevertheless, some multispan helical transmembrane proteins have been proposed to partition into these environments. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the helical tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither S-palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for this preference. In contrast, Charcot-Marie-Tooth disease-causing mutations that disrupt the stability of PMP22 tertiary structure reduce or eliminate this preference in favor of the disordered phase. These studies demonstrate that the ordered phase preference of PMP22 derives from global structural features associated with the folded form of this protein, providing a glimpse at the structural factors that promote raft partitioning for multispan helical membrane proteins.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin.

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

Caveolae: The FAQs.

Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round-table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.

The C99 domain of the amyloid precursor protein resides in the disordered membrane phase.

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by ß-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-ß (Aß) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, ∼90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yet-unidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.

Emerging Insights into the Molecular Architecture of Caveolin-1.

Caveolins are an unusual family of membrane proteins whose primary biological function is to build small invaginated membrane structures at the surface of cells known as caveolae. Caveolins and caveolae regulate numerous signaling pathways, lipid homeostasis, intracellular transport, cell adhesion, and cell migration. They also serve as sensors and protect the plasma membrane from mechanical stress. Despite their many important functions, the molecular basis for how these 50-100 nm "little caves" are assembled and regulate cell physiology has perplexed researchers for 70 years. One major impediment to progress has been the lack of information about the structure of caveolin complexes that serve as building blocks for the assembly of caveolae. Excitingly, recent advances have finally begun to shed light on this long-standing question. In this review, we highlight new developments in our understanding of the structure of caveolin oligomers, including the landmark discovery of the molecular architecture of caveolin-1 complexes using cryo-electron microscopy.

A novel computational framework for D(t) from Fluorescence Recovery after Photobleaching data reveals various anomalous diffusion types in live cell membranes.

Diffusion of proteins and lipids in lipid membranes plays a pivotal role in almost all aspects of cellular biology, including motility, exo-/endocytosis and signal transduction. For this reason, gaining a detailed understanding of membrane structure and function has long been a major area of cell biology research. To better elucidate this structure-function relationship, various tools have been developed for diffusion measurements, including Fluorescence Recovery After Photobleaching (FRAP). Because of the complexity of cellular microenvironments, biological diffusion is often correlated over time and described by a time-dependent diffusion coefficient, D(t), although the underlying mechanisms are not fully understood. Since D(t) provides important information regarding cellular structures, such as the existence of subresolution barriers to diffusion, many efforts have been made to quantify D(t) by FRAP assuming a single power law, D(t) = Γt α - 1 where Γ and α are transport coefficient and anomalous exponent. However, straightforward approaches to quantify a general form of D(t) are lacking. In this study, we develop a novel mathematical and computational framework to compute the mean square displacement of diffusing molecules and diffusion coefficient D(t) from each individual time point of confocal FRAP data without the single power law assumption. Additionally, we developed an auxiliary equation for D(t) which can readily distinguish normal diffusion or single power law anomalous diffusion from other types of anomalous diffusion directly from FRAP data. Importantly, by applying this approach to FRAP data from a variety of membrane markers, we demonstrate the single power law anomalous diffusion assumption is not sufficient to describe various types of D(t) of membrane proteins. Lastly, we discuss how our new approaches can be applied to other fluorescence microscopy tools such as Fluorescence Correlation Spectroscopy (FCS) and Single Particle Tracking (SPT).

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

Helicobacter pylori VacA is a secreted pore-forming toxin that induces cell vacuolation in vitro and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NH4Cl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the hypothesis that NH4Cl augments VacA toxicity by altering the intracellular trafficking of VacA or inhibiting intracellular VacA degradation. We observed VacA colocalization with LAMP1- and LC3-positive vesicles in both the presence and absence of NH4Cl, indicating that NH4Cl does not alter VacA trafficking to lysosomes or autophagosomes. Conversely, we found that supplemental NH4Cl significantly increases the intracellular stability of VacA. By conducting experiments using chemical inhibitors, stable ATG5 knockdown cell lines, and ATG16L1 knockout cells (generated using CRISPR/Cas9), we show that VacA degradation is independent of autophagy and proteasome activity but dependent on lysosomal acidification. We conclude that weak bases like ammonia, potentially generated during H. pylori infection by urease and other enzymes, enhance VacA toxicity by inhibiting toxin degradation.

Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.

MAP1LC3B (microtubule-associated protein 1 light chain 3, LC3) is a key component of the autophagy pathway, contributing to both cargo selection and autophagosome formation in the cytoplasm. Emerging evidence suggests that nuclear forms of LC3 are also functionally important; however, the mechanisms that facilitate the nuclear targeting and trafficking of LC3 between the nucleus and cytoplasm under steady-state conditions are poorly understood. In this study, we examine how residues known to regulate the interactions between LC3 and other proteins or RNA (F52 L53, R68-R70 and G120) contribute to its nuclear targeting, nucleocytoplasmic transport and association with nucleoli and other nuclear components. We find that residues F52 L53 and R68-70, but not G120, regulate targeting of LC3 to the nucleus, its rates of nucleocytoplasmic transport and the apparent sizes of LC3-associated complexes in the nucleus inferred from fluorescence recovery after photobleaching (FRAP) measurements. We also show that LC3 is enriched in nucleoli and its triple arginine motif is especially important for nucleolar targeting. Finally, we identify a series of candidate nuclear LC3-interacting proteins using mass spectrometry, including MAP1B, tubulin and several 40S ribosomal proteins. These findings suggest LC3 is retained in the nucleus in association with high-molecular weight complexes that continuously scan the nucleolus.

Characterization of a caveolin-1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin-1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C-terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae-associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C-terminus of wild-type CAV1 in caveolae, reduced colocalization of cavin-1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 -/- mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.

Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA.

Helicobacter pylori, a Gram-negative bacterium, is a well-known risk factor for gastric cancer. H. pylori vacuolating cytotoxin A (VacA) is a secreted pore-forming toxin that induces a wide range of cellular responses. Like many other bacterial toxins, VacA has been hypothesized to utilize lipid rafts to gain entry into host cells. Here, we used giant plasma membrane vesicles (GPMVs) as a model system to understand the preferential partitioning of VacA into lipid rafts. We show that a wild-type (WT) toxin predominantly associates with the raft phase. Acid activation of VacA enhances binding of the toxin to GPMVs but is not required for raft partitioning. VacA mutant proteins with alterations at the amino terminus (resulting in impaired membrane channel formation) and a nonoligomerizing VacA mutant protein retain the ability to preferentially associate with lipid rafts. Consistent with these results, the isolated VacA p55 domain was capable of binding to lipid rafts. We conclude that the affinity of VacA for rafts is independent of its capacity to oligomerize or form membrane channels.

Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.

Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.

Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Caveolin-1 (Cav1) is the primary scaffolding protein of caveolae, flask-shaped invaginations of the plasma membrane thought to function in endocytosis, mechanotransduction, signaling and lipid homeostasis. A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins. However, how different tags affect the behavior of ectopically expressed Cav1 is still largely unknown. To address this question, we performed a comparative analysis of the subcellular distribution, oligomerization state and detergent resistance of transiently overexpressed Cav1 labeled with three different C-terminal tags (EGFP, mCherry and myc). We show that addition of fluorescent protein tags enhances the aggregation and/or degradation of both wild-type Cav1 and an oligomerization defective P132L mutant. Strikingly, complexes formed by overexpressed Cav1 fusion proteins excluded endogenous Cav1 and Cav2, and the properties of native caveolins were largely preserved even when abnormal aggregates were present in cells. These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae. They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.