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Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases., (© 2021. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Cell division without growth results in progressive cell size reductions during early embryonic development. How do the sizes of intracellular structures and organelles scale with cell size and what are the functional implications of such scaling relationships? Model organisms, in particular Caenorhabditis elegans worms, Drosophila melanogaster flies, Xenopus laevis frogs, and Mus musculus mice, have provided insights into developmental size scaling of the nucleus, mitotic spindle, and chromosomes. Nuclear size is regulated by nucleocytoplasmic transport, nuclear envelope proteins, and the cytoskeleton. Regulators of microtubule dynamics and chromatin compaction modulate spindle and mitotic chromosome size scaling, respectively. Developmental scaling relationships for membrane-bound organelles, like the endoplasmic reticulum, Golgi, mitochondria, and lysosomes, have been less studied, although new imaging approaches promise to rectify this deficiency. While models that invoke limiting components and dynamic regulation of assembly and disassembly can account for some size scaling relationships in early embryos, it will be exciting to investigate the contribution of newer concepts in cell biology such as phase separation and interorganellar contacts. With a growing understanding of the underlying mechanisms of organelle size scaling, future studies promise to uncover the significance of proper scaling for cell function and embryonic development, as well as how aberrant scaling contributes to disease. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Early Embryonic Development > Fertilization to Gastrulation Comparative Development and Evolution > Model Systems., (© 2020 Wiley Periodicals, Inc.)

Zygosaccharomyces rouxii is an important yeast that is required in the food fermentation process due to its high salt tolerance. In this study, the responses and resistance strategies of Z. rouxii against salt stress were investigated by performing physiological analysis at membrane level. The results showed that under salt stress, cell integrity was destroyed, and the cell wall was ruptured, which was accompanied by intracellular substance spillover. With an increase of salt concentrations, intracellular Na + content increased slightly, whereas intracellular K + content decreased significantly, which caused the increase of the intracellular Na + /K + ratio. In addition, in response to salt stress, the activity of Na + /K + -ATPase increased from 0.54 to 2.14 μmol/mg protein, and the ergosterol content increased to 2.42-fold to maintain membrane stability. Analysis of cell membrane fluidity and fatty acid composition showed that cell membrane fluidity decreased and unsaturated fatty acid proportions increased, leading to a 101.21% rise in the unsaturated/saturated fatty acid ratio. The results presented in this study offer guidance in understanding the salt tolerance mechanism of Z. rouxii , and in developing new strategies to increase the industrial utilization of this species under salt stress.

Membrane blebbing accompanies various cellular processes, including cytokinesis, apoptosis, and cell migration, especially invasive migration of cancer cells. Blebs are extruded by intracellular pressure and are initially cytoskeleton-free, but they subsequently assemble the cytoskeleton, which can drive bleb retraction. Despite increasing appreciation of physiological significance of blebbing, the molecular and, especially, structural mechanisms controlling bleb dynamics are incompletely understood. We induced membrane blebbing in human HT1080 fibrosarcoma cells by inhibiting the Arp2/3 complex. Using correlative platinum replica electron microscopy, we characterize cytoskeletal architecture of the actin cortex in cells during initiation of blebbing and in blebs at different stages of their expansion-retraction cycle. The transition to blebbing in these conditions occurred through an intermediate filopodial stage, whereas bleb initiation was biased toward filopodial bases, where the cytoskeleton exhibited local weaknesses. Different stages of the bleb life cycle (expansion, pausing, and retraction) are characterized by specific features of cytoskeleton organization that provide implications about mechanisms of cytoskeleton assembly and bleb retraction., (© 2019 Chikina et al.)

Blebs are involved in various biological processes such as cell migration, cytokinesis, and apoptosis. While the expansion of blebs is largely an intracellular pressure-driven process, the retraction of blebs is believed to be driven by RhoA activation that leads to the reassembly of the actomyosin cortex at the bleb membrane. However, it is still poorly understood how RhoA is activated at the bleb membrane. Here, we provide evidence demonstrating that myosin II-interacting guanine nucleotide exchange factor (MYOGEF) is implicated in bleb retraction via stimulating RhoA activation and the reassembly of an actomyosin network at the bleb membrane during bleb retraction. Interaction of MYOGEF with ezrin, a well-known regulator of bleb retraction, is required for MYOGEF localization to retracting blebs. Notably, knockout of MYOGEF or ezrin not only disrupts RhoA activation at the bleb membrane, but also interferes with nonmuscle myosin II localization and activation, as well as actin polymerization in retracting blebs. Importantly, MYOGEF knockout slows down bleb retraction. We propose that ezrin interacts with MYOGEF and recruits it to retracting blebs, where MYOGEF activates RhoA and promotes the reassembly of the cortical actomyosin network at the bleb membrane, thus contributing to the regulation of bleb retraction., (© 2018 Jiao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Small membranous vesicles are small closed fragments of membrane. They are released from multivesicular bodies (exosomes) or shed from the surface membrane (microvesicles). They contains various bioactive molecules and their molecular composition varies depending on their cellular origin. Small membranous vesicles have been identified in snake venoms, but the origin of these small membranous vesicles in the venom is controversial. The aim of this study was to verify the origin of the small membranous vesicles in venom of Crotalus durissus terrificus by morphological analyses using electron microscopy. In addition, the protein composition of the vesicles was analyzed by using a proteome approach. The small membranous vesicles present in the venom were microvesicles, since they originated from microvilli on the apical membrane of secretory cells. They contained cytoplasmic proteins, and proteins from the plasma membrane, endoplasmic reticulum (ER), and Golgi membrane. The release of microvesicles may be a mechanism to control the size of the cell membrane of the secretory cells after intense exocytosis. Microvesicle components that may have a role in envenoming include ecto-5'-nucleotidase, a cell membrane protein that releases adenosine, and aminopeptidase N, a cell membrane protein that may modulate the action of many peptides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A 2 (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

Osteoclasts in culture are non-transformed cell types that spontaneously develop specific cell-adhesion devices such as podosomes. An individual podosome is a complex network of filamentous actin (F-actin) unit structure that collectively, with other proteins, self-organizes as the sealing zone. Major matrix degradation on apatite seems to proceed under the ruffled-border domain, which is an enclosed extracellular compartment tightly sealed off by this sealing zone. Presently we found that usually the top of finger-like projections of the ruffled border reached toward the plane of the apatite surface, where a shallow degradation of apatite took place. Simultaneously, we obtained several pieces of structural evidence indicating that a specific protrusion referred to as an invasive podosome (invadopodium), which was continuous with podosomes derived from the sealing zone, invaded deeply into apatite matrix and degraded it. The F-actin architecture of the invasive podosome - an active extracellular matrix-degrading, actin-rich cell protrusion - could be distinguished from that of other punctate F-actin structures including the individual podosome, sealing zone, and ruffled border projection. Invasive podosomes contained 2 different F-actin populations, i.e., an interconnected meshwork and a parallel array of bundles. The morphological variability of these protrusions was apparent, having a single cylindrical to lamella-shaped cytoskeletal organization. Our present observations strongly suggest that the degradation of apatite substrate-resorbing osteoclasts appears to have been preceded by the combined appearance of ruffled border and invasive podosomes, and also occurred simultaneously with cell migration during an alternating cycle of resorption and migration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

Poxviruses are enveloped DNA viruses that replicate within the cytoplasm. The first viral structures are crescents and spherical particles, with a lipoprotein membrane bilayer, that are thought to be derived from the ER. We determined that A17, a conserved viral transmembrane protein essential for crescent formation, forms homo-oligomers and shares topological features with cellular reticulon-like proteins. The latter cell proteins promote membrane curvature and contribute to the tubular structure of the ER. When the purified A17 protein was incorporated into liposomes, 25 nm diameter vesicles and tubules formed at low and high A17 concentrations, respectively. In addition, intracellular expression of A17 in the absence of other viral structural proteins transformed the ER into aggregated three-dimensional (3D) tubular networks. We suggest that A17 is a viral reticulon-like protein that contributes to curvature during biogenesis of the poxvirus membrane., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Scanning Electron Microscopy (SEM) and Light Microscopy (LM) of the oral apparatus of Polypedates teraiensis and Hylarana leptoglossa tadpoles revealed a number of morphological and microstructural alterations in the labial tooth rows, jaw sheaths and marginal papillae on exposure to acidic and alkaline pH. These morphological and microstructural alterations observed in the oral apparatus of the tadpole are discussed in the light of available literature. The significance of the study with reference to adverse effects of acidic as well as alkaline pH on oral apparatus of the tadpole is also discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Membrane skeletal networks form a two-dimensional lattice structure beneath erythrocyte membranes. 4.1R-MPP (membrane palmitoylated protein) 1-glycophorin C is one of the basic molecular complexes of the membrane skeleton. An analogous molecular complex, 4.1G-MPP6-cell adhesion molecule 4 (CADM4), is incorporated into the Schmidt-Lanterman incisure (SLI), a truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system. In this review, the dynamic structure of peripheral nerve fibers under stretching conditions is demonstrated using in vivo cryotechnique. The structures of nerve fibers had a beaded appearance, and the heights of SLI circular-truncated cones increased at the narrow sites of nerve fibers under the stretched condition. The height of SLI-truncated cones was lower in 4.1G-deficient nerve fibers than in wild-type nerve fibers. 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. The signal transduction protein, Src, was also involved in the 4.1G-MPP6-CADM4 molecular complex. The phosphorylation of Src was altered by the deletion of 4.1G. Thus, we herein demonstrate a membrane skeletal molecular complex in SLI that has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cells.

Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate the precision release of intravesicular material from cells. Membrane-bound secretory vesicles transiently dock and fuse at the base of porosomes facing the cytosol to expel pressurized intravesicular contents from the cell during secretion. The structure, isolation, composition, and functional reconstitution of the neuronal porosome complex have greatly progressed, providing a molecular understanding of its function in health and disease. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex composed of >500 protein molecules. Membrane proteins compose the porosome complex, making it practically impossible to solve its atomic structure. However, atomic force microscopy and small-angle X-ray solution scattering studies have provided three-dimensional structural details of the native neuronal porosome at sub-nanometer resolution, providing insights into the molecular mechanism of its function. The participation of several porosome proteins previously implicated in neurotransmission and neurological disorders, further attest to the crosstalk between porosome proteins and their coordinated involvement in release of neurotransmitter at the synapse., (© 2015 by the Society for Experimental Biology and Medicine.)

Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential., (© 2015 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.)

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this "ciliary gate" assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment., (© 2015 The Authors.)

The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A recent finding reports that co-stimulation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) and the chemokine receptor 1 (CCR1) triggered formation of membrane nanotubes among bone-marrow-derived mast cells. The co-stimulation was attained using corresponding ligands: IgE binding antigen and macrophage inflammatory protein 1α (MIP1 α), respectively. However, this approach failed to trigger formation of nanotubes among rat basophilic leukemia (RBL) cells due to the lack of CCR1 on the cell surface (Int. Immunol. 2010, 22 (2), 113-128). RBL cells are frequently used as a model for mast cells and are best known for antibody-mediated activation via FcεRI. This work reports the successful formation of membrane nanotubes among RBLs using only one stimulus, a hapten of 2,4-dinitrophenyl (DNP) molecules, which are presented as nanostructures with our designed spatial arrangements. This observation underlines the significance of the local presentation of ligands in the context of impacting the cellular signaling cascades. In the case of RBL, certain DNP nanostructures suppress antigen-induced degranulation and facilitate the rearrangement of the cytoskeleton to form nanotubes. These results demonstrate an important scientific concept; engineered nanostructures enable cellular signaling cascades, where current technologies encounter great difficulties. More importantly, nanotechnology offers a new platform to selectively activate and/or inhibit desired cellular signaling cascades.

Osteoclasts are highly polarized cells from both morphological and functional points of view. Using quick-freeze, rotary-replication methods combined with cell-shearing, we clarified the variability of cytoplasmic surface of the polarized membranes of osteoclasts seeded on apatite. As to the organization of actin filaments and clathrin sheets, we confirmed almost the same ventral membrane specializations of osteoclasts on apatite as seen on glass plates. The organized actin filaments and membrane-associated particles supported the ruffled border membranes. Inside the actin sealing zone, membrane specializations were not always occupied with the ruffled border but also with other types of membranes. Some osteoclasts formed an actin ring but lacked the ruffled border projections. We report a unique and distinctive membrane modification of apatite-attached osteoclasts, i.e., the presence of dense aggregates of membrane-associated particles and related structures not found in the osteoclasts seeded on glass plates. Actin filament polarity in the podosomes was determined by decoration with myosin S1. The actin filament polarity within podosome appears to be oriented predominantly with its barbed ends toward the core, whereas the interconnecting F-actin appears to be mixed oriented. Two different types of clathrin plaques displayed different distributions: clathrin-dependent endocytosis was observed in the ruffled border regions, whereas flat clathrin sheets were found in the leading edge of lamellipodia and near podosomes. The clathrin sheets adhered to the apatite surface tightly on the ventral membranes overlaying the resorption lacunae. All these membrane specializations as mentioned above may indicate the functional variability of osteoclasts seeded on apatite.

Background: Glucose-6-phosphate dehydrogenase deficiency affects over 400 million people worldwide. The hemolytic anemia in G6PD deficiency is usually triggered by oxidative stress, but the mechanism remains uncertain. We have used atomic force microscopy for studying changes in red blood cell membrane and providing new insights on the mechanism., Methods: G6PD activity assay and molecular genetic tests were used for molecular diagnosis. AFM was used to investigate alterations in the ultrastructure of G6PD deficient RBC membranes, the influence of different primaquine concentrations, and the protective effects of vitamin C., Result: Nine variants were identified from 33 G6PD deficient individuals. AFM imaging and quantitative analysis showed that G6PD deficient erythrocytes became heterogeneous and roughness measurements of erythrocyte membranes are increased. G6PD enzyme activity and different mutations may relate with roughness parameters. Furthermore, primaquine induces an increased roughness and height of erythrocyte membrane. Meanwhile, primaquine induces damages to erythrocytes which could be prevented by vitamin C treatment in normal RBCs but not in G6PD deficient erythrocytes., Conclusions: Our research may give valuable information about the status of G6PD deficient patients and explore the mechanism of hemolytic anemia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Biological membranes with cubic symmetry are a hallmark of virus-infected or diseased cells. The mechanisms of formation and specific cellular functions of cubic membranes, however, are unclear. The best-documented cubic membrane formation occurs in the free-living giant amoeba Chaos carolinense. In that system, mitochondrial inner membranes undergo a reversible structural change from tubular to cubic membrane organization upon starvation of the organism. As a prerequisite to further analyze the structural and functional features of cubic membranes, we adapted protocols for the isolation of mitochondria from starved amoeba and have identified buffer conditions that preserve cubic membrane morphology in vitro. The requirement for high concentration of ion-chelating agents in the isolation media supports the importance of a balanced ion milieu in establishing and maintaining cubic membranes in vivo.

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca(2+) concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca(2+) concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca(2+) concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

Essential trace elements Ni(2+) and Cu(2+) can block pollen germination without causing cell death. Mechanisms of this effect remain unclear. Using TEM, we studied the effects of Ni(2+) or Cu(2+) treatment on the ultrastructure of the aperture regions in tobacco pollen preparing to germinate in vitro, since in these zones, the main fluxes of water, ions, and metabolites cross the plasmalemma. Neither Ni(2+) nor Cu(2+) altered the cytoplasm ultrastructure, but both affected the reorganization of apertural periplasm during pollen activation. Numerous multilamellar membranous structures continuous with the plasma membrane could be seen in hydrated but not yet activated pollen. When the normal activation was completed, the structures disappeared and the plasmalemma became smooth. In the presence of 1 mM Ni(2+) or 100 μM Cu(2+), these structures preserved its original appearance. It is assumed to be the storage form for the membrane material, which is to provide an initial phase of the pollen tube growth. Ni(2+) and Cu(2+) affect the utilization of these membranes, thereby, blocking the pollen germination.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Outer membrane vesicles (OMVs) derived from the alphaproteobacterium Ahrensia kielensis and from Pseudoalteromonas marina, a gammaproteobacterium, were sampled from liquid cultures in order to extract the MV-associated DNA, establish a shotgun library, and sequence randomly chosen clones to determine the origins of their DNA. We show that OMVs from A. kielensis and from P. marina both harbour DNA larger than 20 or 30 kbp. Transmission electron microscopical inspection of OMVs of A. kielensis and P. marina showed two types of vesicles: bilayered OMVs with a diameter between 30 and 250 nm and double bilayered OMVs ranging between 80 and 200 nm. Bilayered OMVs are either characterized by the presence of a large electron-dense substance or are elctron translucent. Double bilayered OMVs contained an electron dense substance in the core region surrounded by the second bilayer. 30,094 bp of the genome from OMV of A. kielensis and 45,981 bp of that from P. marina were sequenced. The results indicated that all sequences were single copy and that all sequences, with one exception, were similar to prokaryotic sequences, inserted viral sequences were not detected., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Spermatozoa of the plains mouse (Pseudomys australis), like those of most Australian old endemic rodents, contain, in addition to an apical hook, two further processes that extend from the upper concave surface of the head, the ventral processes. This study shows that these processes contain thiol-rich cytoskeletal proteins, which presumably help to maintain their rigidity during sperm transport, together with the overlying cell membrane having abundant intramembranous proteins. To determine the possible functional significance of these processes, an in vitro study of spermatozoon-zona binding was undertaken. The findings suggest that initial sperm binding occurs by way of the cell membrane over the acrosome of the apical hook and that, subsequently, the lateral surfaces of the ventral processes also become tightly bound to the zona matrix. These ventral processes may therefore have evolved to increase sperm adhesion to the outer zona surface and/or to enhance stabilisation of the spermatozoon at the time of zona binding and initial penetration of the egg coat.

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Background: Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h., Results: We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2-3 μm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young's Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton., Conclusions: Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.

The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease., Biological Significance: In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease., (Copyright © 2013. Published by Elsevier B.V.)

Microscopic analysis is a well-accepted technique in microbiology to characterize a single colony and single cell morphotypes. Although colony morphotype can be imaged by light microscopy, morphotypes of microbial cells are normally viewed by transmission electron microscopy (TEM). The specific location of proteins on microbial cell surface or inside the cells is normally detected using immunoelectron microscopy (IEM). Here we describe the technique of detection of type-specific cellular appendages that are produced by several strains of Pseudomonas aeruginosa under conditions of phosphate limitation. The ability to produce the appendages in P. aeruginosa herein described is thus far attributable only to certain multidrug-resistant strains isolated from critically ill patients. The appearance of appendages is highly suppressed in phosphate rich media and enhanced during growth in phosphate depleted media. Under these dual conditions, the absence or presence of appendages correlates with an adhesive and virulent phenotype in P. aeruginosa strains. The proper technique for appendage visualization is critical to facilitate the necessary studies to further elucidate their structure and function.

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered "Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum" were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm(3) volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm(3)) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm(3)). Reflecting the contributions of thousands of pigment-pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000-2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.

The A3-adenosine receptor (A3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A3AR ligand, we show that A3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A3ARs promote the formation of filipodia-like projections (cytonemes) that can extend up to 100 μm to tether and 'reel in' pathogens. Exposure to bacteria or an A3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A3AR-selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A3AR as a potential target for modulating their function.

In this work the properties of monomolecular films composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) and cholesterol, differing in lipid proportion, were investigated in the context of domain formation in the inner leaflet of membrane. To perform comprehensive analysis of the studied model systems the Langmuir monolayer experiments were performed in combination with Brewster angle microscopy (BAM) and Grazing incidence X-ray diffraction (GIXD) techniques. The analysis of the collected data proved non-ideal behavior of the investigated films. It was found that cholesterol at its lower concentration in the system (10%) is of disturbing influence on SOPE film. Further addition of cholesterol into phospholipids film (33, 50, and 67% of cholesterol) induces an ordering effect on SOPE acyl chains and provokes the formation of sterol-poor and sterol-rich domains which stoichiometry is independent of monolayer composition. The foregoing findings allow one to conclude that in cytosolic leaflet of membrane the lipids may segregate into domains of various cholesterol contents which depending on their composition may play different roles in membrane functioning., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4B (C74A) mutant and atg4b (-/-) bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.

Tunneling nanotubes (TNTs) and associated structures are recently recognized structures for intercellular communication. They are F-actin-containing thin protrusions of the plasma membrane of a cell and allow a direct physical connection to the plasma membranes of remote cells. TNTs and associated structures serve as mediators for intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, several pathogens have been shown to exploit these structures to spread among cells. Because of their contribution to normal cellular functions and importance in pathological conditions, studies on TNTs and related structures have accelerated over the past few years. These studies have revealed key molecules for their induction and/or formation; HIV Nef and M-Sec can induce the formation of TNTs in coordination with the remodeling of the actin cytoskeleton and vesicle trafficking.

CI-976 is a lysophospholipid acyltransferase antagonist that is known to affect secretory and endocytic membrane-trafficking pathways likely by increasing the lysophospholipid content in membranes. Our previous study suggested that lysophospholipids formed through the action of an intracellular phospholipase A(1), KIAA0725p (also known as DDHD2 and iPLA(1)γ), may be important for the association of this enzyme with membranes. In this study, we examined the effect of CI-976 on the membrane association of KIAA0725p. While in HeLa cells KIAA0725p is localized in the Golgi and cytosol, in mouse embryonic fibroblasts (MEFs), it was found to be principally localized in the cytosol with some on post-endoplasmic reticulum compartments including the cis-Golgi. Treatment of MEFs with CI-976 induced the redistribution of KIAA0725p to membrane tubules, which were in vicinity to fragmented mitochondria. These tubules were not decorated with canonical organelle markers including Golgi proteins. A human KIAA0725p mutant, which exhibits decreased membrane-binding ability, was also redistributed to membrane structures upon CI-976 treatment. Our data suggest that the association of KIAA0725p with membranes is regulated by lipid metabolism, and that CI-976 may create unique membrane structures that can be marked by KIAA0725p.

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle., (© 2012 Blackwell Publishing Ltd.)

Red blood cells (RBCs) are highly deformable and possess a robust membrane that can withstand shear force. Previous research showed that in diabetic patients, there is a changed RBC ultrastructure, where these cells are elongated and twist around spontaneously formed fibrin fibers. These changes may impact erythrocyte function. Ultrastructural analysis of RBCs in inflammatory and degenerative diseases can no longer be ignored and should form a fundamental research tool in clinical studies. Consequently, we investigated the membrane roughness and ultrastructural changes in type 2 diabetes. Atomic force microscopy (AFM) was used to study membrane roughness and we correlate this with scanning electron microscopy (SEM) to compare results of both the techniques with the RBCs of healthy individuals. We show that the combined AFM and SEM analyses of RBCs give valuable information about the disease status of patients with diabetes. Effectiveness of treatment regimes on the integrity, cell shape and roughness of RBCs may be tracked, as this cell's health status is crucial to the overall wellness of the diabetic patient.

Gram-positive bacteria can transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here, we develop and analyse a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range of 15-24 nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insights into the range of cell wall hole sizes compatible with bacterial homeostasis.

Atomic force microscopy (AFM) allows a researcher to obtain images of red blood cells (RBC) and their membranes. Various effects on blood lead to surface alterations of cell membranes. Such alterations are estimated by a corrugation of membrane surface. This problem is complicated for statistical analysis because the membrane is the ensemble of structures with different sizes. In the present work we used the space Fourier transform to decompose the complex AFM image of the surface into three simpler ones. The parameters of spectral windows were selected according to the natural structures of RBC membranes. This method allowed us to obtain high resolution images for the corresponding spectral windows, to establish specificity of alterations from each effect, to estimate quantitatively the membrane nanostructures at different space scales and to compare their sizes statistically after actions of different agents. The blood intoxication was modeled by adding hemin, furosemide, chlorpromazine and zinc ions into blood, in vitro., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities. We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes. To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation. Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

In human monocyte-derived macrophages (MDM), human immunodeficiency virus type 1 (HIV-1) assembly takes place primarily on complex intracellular plasma membrane domains connected to the cell surface by closely apposed membrane sheets or narrow channels. Some of the membranes associated with these compartments are decorated by thick (≈30 nm), electron-dense, cytoplasmic coats. Here we show by immunolabelling of ultrathin cryosections that the β2 integrin CD18, together with the αM and αX integrins (CD11b and CD11c), is clustered at these coated domains, and that the coats themselves contain the cytoskeletal linker proteins talin, vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton. Intracellular plasma membrane-connected compartments (IPMC) with CD18-containing focal adhesion-like coats are also present in uninfected MDM. These compartments become more prominent as the cells mature in tissue culture and their appearance correlates with increased expression of CD18, CD11b/c and paxillin. Depletion of CD18 by RNA interference leads to parallel down-regulation of CD11b and CD11c, as well as of paxillin, and the disappearance of the adhesion-like coats. In addition, CD18 knockdown alters the appearance of virus-containing IPMC in HIV-infected MDM, indicating that the β2 integrin/focal adhesion-like coat structures are involved in the organization of these compartments., (© 2011 John Wiley & Sons A/S.)