Search

Background Information Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS‐CoV‐2 isolate from São Paulo state (Brazil). Results Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. Conclusions and Significance This study contributes to a better understanding of the cell biology of SARS‐CoV‐2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus‐induced membrane remodelling., Research article: Schematic view of the route of nascent severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) through the compartments of the viral factory. SARS‐CoV‐2 infection induces an intense intracellular membrane remodelling in Vero cells. This alteration protects and delimits the locus of viral replication, morphogenesis and maturation. The intermediate steps of SARS‐CoV‐2 morphogenesis were approached by transmission electron microscopy and electron tomography, revealing and describing different membrane structures and the assembly of viral particles by budding through the endoplasmic reticulum‐Golgi intermediate compartment.

Extracellular vesicles (EVs), a broad term for the lipid microparticles known as microvesicles and exosomes, are discharged by cells into their surrounding space. Microvesicles are discharged upon outward plasma membrane budding, while exosomes are secreted after multivesicular body (MVB) fusion with the plasma membrane. The majority of information regarding EV biology comes from studies performed in non-polarized cells. Here we characterize EV release in polarized cells. We found a substantial asymmetry in the number and composition of EVs produced and released from the apical membrane of epithelial cells as compared to the basolateral membrane. We showed that the quantitative difference is related to the polarized distribution of two phosphoinositide species between the two cell surfaces and that the peculiar biochemical composition of resultant EVs reflects their site of origin. In particular, apical and basolateral exosomes may derive from distinct classes of MVBs originating from and fusing with the same plasma membrane. We identify VAMP8/Endobrevin as a regulator of the basolateral release of exosomes, whereas the mechanism responsible for apical EV release requires further study.

Shrub (CG8055) encodes the vps32/snf7 protein, a filament-forming subunit of the ESCRT (endosomal sorting complexes required for transport)-III complex involved in inward membrane budding. It was reported that shrub was required for abscission in female germline stem cells. In this study, we showed that the expression level of shrub in the testis was significantly higher than that in the ovary of 1-day-old Drosophila melanogaster, suggesting a role in male reproduction. Then we used nosGal4 driver to knockdown shrub specifically in the fly testis and found that this resulted in a significantly lower paternal effect egg hatch rate relative to the control group. Immunofluorescence staining showed that shrub knockdown in fly testes caused an accumulation of early-stage germ cells and lack of spectrin caps. In the late stages (spermiogenesis), the control testis contained multiple compacted spermatid bundles and individualization complexes (ICs) consisting of actin cones, whereas there were scattered spermatid nuclei and only a few ICs with disorganized actin cones in the shrub knockdown testis. Finally, the control seminal vesicle was full of mature sperms with needle-like heads, but in shrub knockdown testis 75% of seminal vesicles had no mature sperms. We also found that knockdown of shrub in fly testes led to upregulated expression of several cytoskeleton-associated genes, and an accumulation of ubiquitylated proteins. These results suggest that knockdown of shrub in fly testes might damage spermatogenesis by affecting transportability.

Complex molecular machineries bud, scission and repair cellular membranes. Components of the multi-subunit endosomal sorting complex required for transport (ESCRT) machinery are enlisted when multivesicular bodies are generated, extracellular vesicles are formed, the plasma membrane needs to be repaired, enveloped viruses bud out of host cells, defective nuclear pores have to be cleared, the nuclear envelope must be resealed after mitosis and for final midbody abscission during cytokinesis. While some ESCRT components are only required for specific processes, the assembly of ESCRT-III polymers on target membranes and the action of the AAA-ATPase Vps4 are mandatory for every process. In this review, we summarize the current knowledge of structural and functional features of ESCRT-III/Vps4 assemblies in the growing pantheon of ESCRT-dependent pathways. We describe specific recruitment processes for ESCRT-III to different membranes, which could be useful to selectively inhibit ESCRT function during specific processes, while not affecting other ESCRT-dependent processes. Finally, we speculate how ESCRT-III and Vps4 might function together and highlight how the characterization of their precise spatiotemporal organization will improve our understanding of ESCRT-mediated membrane budding and scission in vivo.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells – nucleic acids, proteins, lipids – are possibly modified during the process. Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres. The results of our comparisons and the failure to generate mutants lacking the ability to convert to spheres suggest that the metamorphosis of B. burgdorferi results in a conditional reconstruction of the outer membrane. The spheres, which appear to be more resistant to unfavorable conditions and exhibit reduced immune reactivity when compared to spirochetes, might allow the B. burgdorferi to escape complete clearance and possibly ensure long-term survival in the host. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Phosphoinositides are a family of minority acidic phospholipids in cell membranes. Their principal role is instructional: they interact with proteins. Each cellular membrane compartment uses a characteristic species of phosphoinositide. This signature phosphoinositide attracts a specific complement of functionally important, loosely attached peripheral proteins to that membrane. For example, the phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane attracts phospholipase C, protein kinase C, proteins involved in membrane budding and fusion, proteins regulating the actin cytoskeleton, and others. Phosphoinositides also regulate the activity level of the integral membrane proteins. Many ion channels of the plasma membrane need the plasma-membrane-specific PIP2 to function. Their activity decreases when the abundance of this lipid falls, as for example after activation of phospholipase C. This behaviour is illustrated by the suppression of KCNQ K+ channel current by activation of M1 muscarinic receptors; KCNQ channels require PIP2 for their activity. In summary, phosphoinositides contribute to the selection of peripheral proteins for each membrane and regulate the activity of the integral proteins.

Yeast Dop1p is an essential protein that is highly conserved in evolution and whose function is largely unknown. Here, we provide evidence that Dop1p localizes to endosomes and exists in a complex with two other conserved proteins: Neo1p, a P(4)-ATPase and putative flippase, and the scaffolding protein Ysl2p/Mon2p. The latter operates during membrane budding at the tubular endosomal network/trans-Golgi network (TEN/TGN) in a process that includes clathrin recruitment via adaptor proteins. Consistent with a role for Dop1p during this process, temperature-sensitive dop1-3 cells accumulate multivesicular, elongated tubular and ring-like structures similar to those displayed by neo1 and ysl2 mutants. In further agreement with the concept of Dop1p-Neo1p-Ysl2p complex formation and co-operation, we show that dop1-3 cells exhibit reduced levels of Neo1p and Ysl2p at steady state. Conversely, mutations or deletions in NEO1 and YSL2 lead to a decrease in Dop1p levels. In addition to binding to Neo1p and Ysl2p, Dop1p can form dimers or multimers. A critical region for dimerization resides in the C-terminus with leucine zipper-like domains. Dop1p's membrane association is largely mediated by its internal region, but Ysl2p might not be crucial for membrane recruitment.

The aim of this study was to examine the neurotoxicity of formaldehyde on prefrontal cortex and the protective effects of omega-3 essential fatty acids against these toxic effects. For this purpose, 21 male Wistar rats were divided into three groups. The rats in group I comprised the controls, while the rats in group II were injected every other day with formaldehyde (FA). The rats in group III received omega-3 fatty acids daily while exposed to formaldehyde. At the end of the 14-day experimental period, all rats were killed by decapitation. The brains of the rats were removed and the prefrontal cortex tissues were obtained from all brain specimens. Some of the prefrontal cortex tissue specimens were used for determination of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels. The remaining prefrontal cortex tissue specimens were used for light microscopic and immunohistochemical evaluation. The levels of SOD and GSH-Px were significantly decreased, and MDA levels were significantly increased in rats treated with formaldehyde compared with those of the controls. Furthermore, in the microscopic examination of this group, formation of apoptotic bodies, pycnotic cells, and apoptotic cells including nuclear fragmentation and membrane budding were observed. However, increased SOD and GSH-Px enzyme activities, and decreased MDA levels were detected in the rats administered omega-3 fatty acids while exposed to formaldehyde. Additionally, cellular damage caused by formaldehyde was decreased, and structural appearance was similar to that of the control rats in this group. The biochemical and histological findings observed in all groups were also confirmed by immunohistochemical evaluation. It was determined that formaldehyde-induced neuronal damage in prefrontal cortex was prevented by administration of omega-3 essential fatty acids.

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

The sections in this article are: 1 Intracellular Transport in Vesicles 2 Membrane Fusion and Exocytosis 2.1 Investigations in Synaptic Vesicles 2.2 Investigations in Yeast 2.3 Reconstituted Systems of Transport 2.4 Convergence of Separate Approaches 2.5 Calcium Dependence of Stimulated Neurotransmitter Release 2.6 Docking Vesicles to the Correct Membrane 2.7 Other Fusion Mechanisms 2.8 Evidence for SNAP-NSF-Mediated Docking/Fusion With Dense Core Granules 2.9 Exocytosis in Endocrine Cells 3 Membrane Budding and Endocytosis 3.1 Budding Involving Clathrin 3.2 Budding Involving COPI/ARF 3.3 Endocytosis in Endocrine Cells 3.4 Formation of Secretory Granules in Endocrine Cells

Brefeldin A induces a rapid redistribution of coat proteins associated with clathrin-coated vesicles that bud from the trans-Golgi network but not of those associated with vesicles derived from the plasma membrane. Its effect on glucose transport and the subcellular distribution of the glucose transporter GLUT4 were therefore investigated in the absence or in the presence of insulin in order to characterize the mechanism of insulin-induced GLUT4 recycling. Exposure of rat adipose cells to brefeldin A in the absence of insulin resulted in a slight increase both in 3-O-methy-d-glucose transport and in the amount of GLUT4 in the plasma membrane as determined by immunoblot analysis. In contrast, it had no effect on the insulin-induced increase in 3-O-methyl-glucose transport and plasma-membrane-associated GLUT4. Immunohistochemical analysis revealed clathrin immunoreactivity in the juxtanuclear region and in the cell periphery, and the staining in the perinuclear region decreased after exposure of cells to 35 μM brefeldin A. However, it had no effect on the basal pattern of GLUT4 staining or on the insulin-induced redistribution of GLUT4 staining to the cell periphery. These results suggest that basal recycling of GLUT4 responsible for maintaining the intracellular location of the transporter may include a membrane budding step sensitive to brefeldin A, possibly associated with clathrin-coated vesicles, but that the insulin-stimulated GLUT4 recycling pathway is mediated predominantly by a brefeldin A-insensitive mechanism.

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-terminal additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.

The rapid movement of polar lipids from one membrane leaflet to the other is facilitated by lipid flippases or translocases. Although their activity was first observed over 30 years ago, the structures, physiological roles, and molecular mechanisms of this group of proteins remain enigmatic. Lipid flippases maintain membrane lipid asymmetry, and in eukaryotes they are also intimately involved in membrane budding and vesicle trafficking. The ATP-dependent flippases are members of well-characterized protein families, whose other members transport nonlipid substrates across cell membranes. The P(4)-type ATPases carry out the inward translocation of phospholipids, and various ABC transporters are involved in outward lipid movement. The ATP-independent flippases move lipid substrates in both directions between membrane leaflets. With only a few exceptions, the molecular identity of these proteins is still unknown, despite their involvement in key biosynthetic pathways in both bacteria and eukaryotes. This review provides an overview of the different classes of flippases, and summarizes recent progress in their identification and functional characterization. The possible mechanisms of action of lipid flippases are discussed, and future directions explored.

There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.