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Late endosome and lysosomes form the second major membrane network along the endocytic pathway, after early/recycling endosomes, and as such, late endosomes function as the last portal before lysosomes. In mammalian cells, they also link the endosomal system to other cellular destinations, including the trans-Golgi complex and the plasma membrane. Finally it has become clear in recent years that late endosomes operate as a major sensing and signaling organelle that informs the cell about its nutrient and supply situation.

Plasmodium merozoites invade erythrocytes in a stepwise manner through ligand binding, calcium signaling, and membrane deformation. Using a recently developed light-sheet microscope, Geoghegan et al. investigated invasion with unprecedented temporal resolution. Their spectacular footage revealed roles for host cell cholesterol and pore formation at the parasite-host cell interface.

New work describes a novel mechanism of mechanotransduction, whereby force-induced membrane deformation activates integrins by disrupting the association of the transmembrane domains of α and β integrins.

Depending on the physical and biochemical properties of actin-binding proteins, actin networks form different types of membrane protrusions at the cell periphery. Actin crosslinkers, which facilitate the interaction of actin filaments with one another, are pivotal in determining the mechanical properties and protrusive behavior of actin networks. Short crosslinkers such as fascin bundle F-actin to form rigid spiky filopodial protrusions. By encapsulation of fascin and actin in giant unilamellar vesicles (GUVs), we show that fascin-actin bundles cause various GUV shape changes by forming bundle networks or straight single bundles depending on GUV size and fascin concentration. We also show that the presence of a long crosslinker, α-actinin, impacts fascin-induced GUV shape changes and significantly impairs the formation of filopodia-like protrusions. Actin bundle-induced GUV shape changes are confirmed by light-induced disassembly of actin bundles leading to the reversal of GUV shape. Our study contributes to advancing the design of shape-changing minimal cells for better characterization of the interaction between lipid bilayer membranes and actin cytoskeleton.

Previous studies from our laboratory established that pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex type 2 (HSV-2) capsids. To better understand the role of these proteins in nuclear egress, we wished to establish whether nuclear egress complex (NEC) localization and/or function was altered in the absence of either pUL16 or pUL21. We used antiserum raised against HSV-2 NEC components pUL31 and pUL34 to examine NEC localization by immunofluorescence microscopy. NEC localization in cells infected with pUL16 deficient viruses was indistinguishable from that observed in cells infected with wild type viruses. By contrast, NEC localization was found to be aberrant in cells infected with pUL21 deficient virus and, instead, showed some similarity to the aberrant NEC localization pattern observed in cells infected with pUs3 deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher resolution examination of nuclear envelope ultrastructure in cells infected with pUL21 deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21 deficient virus infections was found to be dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16 or both pUs3 and pUL16 were consistent with a role for pUL16 upstream of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.Author summaryThe membrane deformation activity of the herpesvirus nuclear egress complex (NEC), allows viral capsids to transit from their site of assembly in the nucleus through both nuclear membranes into the cytoplasm. The timing, extent and directionality of NEC activity must be precisely controlled during viral infection, yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus type 2 (HSV-2) capsids, function to promote nuclear egress, we examined how the lack of each protein impacted NEC localization. These analyses revealed a function of pUL16 in nuclear egress that is distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of viral capsids is a common feature of the replicative cycle of all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process that could be applied to the development of herpes-specific antiviral drugs.

The nuclear pore complex (NPC) mediates all macromolecular transport across the nuclear envelope. In higher eukaryotes that have an open mitosis, NPCs assemble at two points in the cell cycle: during nuclear assembly in late mitosis and during nuclear growth in interphase. How the NPC, the largest nonpolymeric protein complex in eukaryotic cells, self-assembles inside cells remained unclear. Recent studies have started to uncover the assembly process, and evidence has been accumulating that postmitotic and interphase NPC assembly use fundamentally different mechanisms; the duration, structural intermediates, and regulation by molecular players are different and different types of membrane deformation are involved. In this Review, we summarize the current understanding of these two modes of NPC assembly and discuss the structural and regulatory steps that might drive the assembly processes. We furthermore integrate understanding of NPC assembly with the mechanisms for rapid nuclear growth in embryos and, finally, speculate on the evolutionary origin of the NPC implied by the presence of two distinct assembly mechanisms.

Epsin N-terminal homology (ENTH) domains are present at the N-terminus of either the epsin or epsin-related (epsinR) proteins. These proteins have been involved in clathrinmediated trafficking and are critical for membrane deformation at the site of vesicle budding. While more than one type of these proteins have been described in many eukaryotic cells, the protozoa parasite Giardia lamblia contains only one member of this ENTH-protein family. In the last two years, four works have been published showing that this giardial protein might play diverse functions. This commentary gives a brief overview on the current status of the particular characteristics and functions of this unique protein. Fil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release. ispartof: MOLECULAR CELL vol:71 issue:5 pages:689-+ ispartof: location:United States status: published

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pβ caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pβ overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pβ was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pβ protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pβ strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1.

Summary Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation., Graphical Abstract, Highlights • FCHSD2 is a bona fide CME protein recruited to CCPs by intersectin • Intersectin recruits FCHSD2 via an SH3-SH3 interaction • FCHSD2 is a major activator of actin during CME • FCHSD2 binds to the surrounding membrane around CCPs via its flat F-BAR domain, A flat BAR protein binds to the membrane surrounding clathrin-coated pits and promotes actin polymerization to aid endocytic vesicle maturation.

Autophagic lysosome reformation (ALR) is the terminal step of autophagy and is essential for maintaining lysosome homeostasis during autophagy. During ALR, tubules are extruded from autolysosomes, and small vesicles named proto-lysosomes, which are composed of lysosomal membrane components, are generated from these tubules. Eventually, proto-lysosomes mature into functional lysosomes. In this review, we will summarize recent progress in understanding the regulation, mechanisms and physiological functions of ALR.

TiO2 nanoparticles (NPs), which are mainly used in consumer products (mostly cosmetics), have been found to cause ecotoxic effects in the aquatic environment. The green algae Raphidocelis subcapitata, as a representative of primary producers of the freshwater ecosystem, has been frequently used to study the effects of metal oxide NPs. An ecotoxicity study was conducted herein to investigate the effects of TiO2 NPs on survival and membrane deformation of algal cells. Five different concentrations of nano-TiO2 particles (1, 10, 50, 100 and 500 mg/L) were prepared in synthetic surface water samples with five different water quality characteristics (pH 6.4-8.4, hardness 10-320 mg CaCO3/L, ionic strength 0.2-8 mM, and alkalinity 10-245 mg CaCO3/L). Results showed a significant increase in the hydrodynamic diameter of NPs with respect to both NP concentrations and ionic content of the test system. A soft synthetic freshwater system at pH 7.3 +/- 0.2 appeared to provide the most effective water type, with more than 95% algal mortality observed at 50, 100 and 500 mg/L NP concentrations. At high exposure concentrations, increased malondialdehyde formations were observed. Moreover, due to membrane deformation, TEM images correlated the uptake of the NPs.

The eukaryotic endomembrane system is responsible for the biosynthesis and transport of proteins and lipids, and for the definition of the major subcellular compartments. Recent work indicates that the endomembrane system is ancient, with near modern complexity predating the radiation of the major eukaryotic lineages. The challenge is to look beyond the last eukaryotic common ancestor and to attempt to deduce the evolutionary steps in the rise of membrane-trafficking complexity. Relationships between the endomembrane coatomer complexes and their evolutionary connection to the nuclear pore complex are emerging. These studies, plus the realization of a role for the ESCRT complex as an alternate, but equally ancient, system for membrane deformation are providing insight into the earliest stages of endomembrane evolution.

Tethering factors have been shown to interact with Rabs and SNAREs and, more recently, with coat proteins. Coat proteins are required for cargo selection and membrane deformation to bud a transport vesicle from a donor compartment. It was once thought that a vesicle must uncoat before it recognizes its target membrane. However, recent findings have revealed a role for the coat in directing a vesicle to its correct intracellular destination. In this review we will discuss the literature that links coat proteins to vesicle targeting events.

Summary ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission., Graphical Abstract, Highlights • Snf7 forms highly flexible filaments that spontaneously curl • Snf7 filaments forms spirals at the surface of lipid membranes • Snf7 spirals are springs as they can deform under lateral compression • Relaxation of compressed Snf7 spirals leads to membrane deformation, A component of the ESCRT-III membrane fission machinery self-organizes into spiral springs that trigger membrane deformation when released.

Clathrin-independent endocytosis removes membrane receptors and other proteins from the cell surface, yet the mechanisms controlling this process remain unclear. Galectin-3 is now shown to regulate the biogenesis of a subpopulation of clathrin-independent carriers (CLICs). Galectin-3 binds to glycosylated cargo proteins and interacts with membrane glycosphingolipids to induce membrane deformation and CLIC formation.

cited By 0 The post-synaptic spines of neuronal dendrites are highly elaborate membrane protrusions. Their anatomy, stability and density are intimately linked to cognitive performance. The morphological transitions of spines are powered by coordinated polymerization of actin filaments against the plasma membrane, but how the membrane-associated polymerization is spatially and temporally regulated has remained ill defined. Here, we discuss our recent findings showing that dendritic spines can be initiated by direct membrane bending by the I-BAR protein MIM/Mtss1. This lipid phosphatidylinositol (PI(4,5)P2) signaling-activated membrane bending coordinated spatial actin assembly and promoted spine formation. From recent advances, we formulate a general model to discuss how spatially concentrated protein-lipid microdomains formed by multivalent interactions between lipids and actin/membrane regulatory proteins might launch cell protrusions. © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC, Pirta Hotulainen and Juha Saarikangas.

Summary Clathrin has been implicated in retromer-mediated trafficking, but its precise function remains elusive. Given the importance of retromers for efficient endosomal sorting, we have sought to clarify the relationship between clathrin and the SNX-BAR retromer. We find that the retromer SNX-BARs do not interact directly or indirectly with clathrin. In addition, we observe that SNX-BAR-retromer tubules and carriers are not clathrin coated. Furthermore, perturbing clathrin function, by overexpressing a dominant-negative clathrin or through suppression of clathrin expression, has no detectable effect on the frequency of SNX-BAR-retromer tubulation. We propose that SNX-BAR-retromer-mediated membrane deformation and carrier formation does not require clathrin, and hence the role of clathrin in SNX-BAR-retromer function would appear to lie in pre-SNX-BAR-retromer cargo sorting.

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 A resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

Transport vesicle coat proteins play active roles in vesicle cargo sorting as well as membrane deformation and fission during vesicle biogenesis. For years, it was assumed that this was the extent of the coats' function and that the coats depolymerized immediately after vesicle budding, leaving the exposed fusion machinery free to find, dock, and fuse with the proper target membrane. Recently, however, it has become increasingly clear that the coat remains on transport vesicles during their post-budding life and in fact helps properly pair up the vesicle with its intended target membrane. These data have brought up urgent questions about exactly when vesicles do uncoat and how uncoating is regulated. Here, we summarize the latest round of evidence for post-budding roles for coats, including a few hints about how the uncoating process may be coupled to docking and fusion. We also speculate about the possibility of post-fusion functions for residual coats.

Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

This paper aims at investigating and characterizing force behavior and mechanical properties of living Drosophila embryos using an in situ modeled PVDF (Polyvinylidene Fluoride) piezoelectric micro-force sensing tool with a resolution in the range of sub-?N. Drosophila embryo is one of the most studied organisms in biological research, medical research, genetics and developmental biology, and has implications in the cure of human diseases. In order to achieve high efficiency and accuracy during microinjection of genetic material into a Drosophila embryo, it is absolutely necessary to allow close monitoring of the magnitude and direction of microinjection forces acting on the embryo during injection. In this paper, a microrobotic biomanipulation platform integrating a two-axis (2-D) modeled PVDF micro-force sensor is used to implement force sensing during microinjection of living Drosophila embryos. Micro injection forces and membrane deformation of embryos in different stages of embryogenesis are found. Ultimately, the technology will provide a critical and major step towards the development of automated biomanipuation for batch microinjection of living embryos in genetics.

Endocytic clathrin-coated vesicles arise through the deformation of a small region of plasma membrane encapsulated by a cytosol-oriented clathrin lattice. The coat assembles from soluble protomers in a rapid and highly cooperative process, and invagination is tightly linked to the selective enrichment of cargo molecules within the nascent bud. Recent structural and functional studies demonstrate that coat assembly, membrane deformation, local actin dynamics and the final scission event are intricately coupled, and begin to reveal how key multifunctional, modular proteins are responsible for this linkage. An emerging mechanistic theme is how sequential engagement of common interaction surfaces or network hubs can evict prior binding partners from the assembly zone to ensure vectorial progression of the coat assembly process.

Single-channel recordings from central neurons of the helix snail, Cepaea nemoralis, revealed two types of channels that could be activated by stretch (i.e., by the membrane deformation produced when suction is applied to the patch pipette). One, a K+ channel (58 pS in physiological solution), was evident in excised and cell-attached patches. Its conductance in symmetrical [K+] solutions indicated a channel of high K+ permeability (PK = 3.4 × 10−13 cm/s). Though osmoregulation has been suggested as a function for such channels, comparisons among molluscs indicate osmotic milieu does not govern their expression; Cepaea is terrestrial, and stretch-activated K+ channels similar to those described here occur in aquatic and marine molluscs. The second type of channel, observed only in excised patches, was Cl− permeant; it had a large conductance (130 pS) and was inactive prior to patch excision. Membrane tension may not be the physiological activator of either the K+ or Cl− channel; the channels are designated as stretch-activated channels on the basis of their experimental behaviour during single-channel recording.Key words: K+ channel, Cl− channel, snail neuron, stretch activation.

Malaria parasites enter red cells in a multi-step process involving attachment, membrane deformation, invagination and encapsulation. The molecular basis of red cell rigidity is examined by Geof f Pasvol and lain Wilson, and they discuss its effect on the efficiency of invasion by various Plasmodium spp.

A scanning electron microscopic examination of antibody-induced hemagglutination of human red cells by an improved enzyme-labeled antibody technique and fixation of the reaction sequence with glutaraldehyde shows that both antigen mobility and membrane deformation are necessary for the reaction to occur.

HER2 overexpression correlates with an increased metastatic potential in breast cancer (BC). While targeted therapies against HER2 effectively delay disease progression in this BC subtype, details of how HER2 overexpression drives these tumors to malignancy are still unclear. To gain a molecular understanding of this process on live cells, we employed quantum dot (QD) based single molecule imaging and analysis methods to monitor spatial arrangements of individual HER2s. Interestingly, overexpressed HER2s were not uniformly distributed on the cell membranes, but observed in clustered and elongated patterns. We found that these patterns resulted from deformed membrane morphologies, which appeared as irregularly shaped ‘finger-like’ structures (FLS) in electron micrograph images of HER2+ BC cells grown in vitro and taken from patients. Quantitative cluster analyses on cells that overexpress signaling-incompetent HER2 mutants showed that this membrane deformation was induced by high HER2 expression rather than by its signaling activities. The membrane deformation reduced cell adhesiveness by disrupting cell-substrate and cell-cell contacts, and perturbed 3D cell organization. These observations suggest that physical alteration of cell membranes by HER2 overexpression can increase the potential for cell dissemination in a non-canonical, signaling-independent manner, which may be involved in invasive progression of HER2+ BC.