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During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The cell membrane structure is closely related to the occurrence and progression of many metabolic bone diseases observed in the clinic and is an important target to the development of therapeutic strategies for these diseases. Strong experimental evidence supports the existence of membrane microdomains in osteoclasts (OCs). However, the potential membrane microdomains and the crucial mechanisms underlying their roles in OCs have not been fully characterized. Membrane microdomain components, such as scaffolding proteins and the actin cytoskeleton, as well as the roles of individual membrane proteins, need to be elucidated. In this review, we discuss the compositions and critical functions of membrane microdomains that determine the biological behavior of OCs through the three main stages of the OC life cycle., (© 2023. The Author(s).)

Importance: Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells., Competing Interests: The authors declare no conflict of interest.

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. We show in Drosophila that the zona pellucida (ZP) domain protein Piopio interacts and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. We also show conserved ectodomain shedding of the human TGFβ type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes., Competing Interests: LD, SS, DR, RS, MB No competing interests declared, (© 2023, Drees et al.)

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gβγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C β3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells use actin-based protrusions not only to migrate, but also to sample their environment and take up liquids and particles, including nutrients, antigens and pathogens. Lamellipodia are sheet-like actin-based protrusions involved in sensing the substratum and directing cell migration. Related structures, macropinocytic cups, arise from lamellipodia ruffles and can take in large gulps of the surrounding medium. How cells regulate the balance between using lamellipodia for migration and macropinocytosis is not yet well understood. We recently identified CYRI proteins as RAC1-binding regulators of the dynamics of lamellipodia and macropinocytic events. This review discusses recent advances in our understanding of how cells regulate the balance between eating and walking by repurposing their actin cytoskeletons in response to environmental cues., (© 2023 The Author(s).)

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell 1-3 . A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated 4-6 , but never visualized. Here we describe a highly enriched culture of 'Candidatus Lokiarchaeum ossiferum', a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7-14 days, reaches cell densities of up to 5 × 10 7 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain 7 . ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying 'Ca. L. ossiferum' cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin-one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures., (© 2022. The Author(s).)

Exogenous melatonin confers the chilling tolerance of banana fruit by promoting reactive oxygen species (ROS) scavenging system and inducing the unsaturated fatty acid synthesis. The results showed that melatonin treatment increased the contents of phospholipids, promoted the ROS scavenging enzyme, and restrained the activities of lipoxygenase (LOX), and thus reduced the lipid peroxidation of banana peel. In addition, melatonin treatment increased the flavonoids and proline contents, which was conducive to antioxidant capacity. Interestingly, the enhanced antioxidant capacity is conducive to the stability of unsaturated fatty acids and reduce the enzymatic browning reaction. Moreover, melatonin treatment induced the expression of omega-3/6 fatty acid desaturase and triggered the fatty acid metabolism activity, by which maintained higher contents of unsaturated fatty acid in banana peel. Moreover, melatonin treatment stimulated the accumulation of fatty acids in banana peel, and was involved in alleviating fruit chilling injury., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ruffles are actin-rich membrane protrusions implicated in actin reorganization and initiation of cell motility. Here, we describe methods for measuring and analyzing ruffle dynamics in live cells and average ruffle area per cell in fixed samples. The specific steps described are for the analysis of A549 lung adenocarcinoma cells, but the protocol can be applied to other cell types. The protocol has applications for dissecting the signaling events linked to ruffling. For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.

Stem cell-based therapy has been indicated to be beneficial for intervertebral disc regeneration. However, the underlying mechanisms have not been fully identified. The present study showed that bone marrow mesenchymal stem cells (BMSCs) donated mitochondria to adjacent nucleus pulposus cells (NPCs) in a coculture system. The mode of mitochondrial transfer between these cells was intercellular tunneling nanotube (TNT), which acted as a transportation expressway for mitochondria. NPCs acquired additional mitochondria from BMSCs in a concentration-dependent manner after rotenone-induced mitochondrial dysfunction in NPCs. Further research demonstrated that TNT-mediated mitochondrial transfer rescued NPCs from mitochondrial dysfunction and apoptosis, which was indicated by the recovery of the mitochondrial respiratory chain, the increase in mitochondrial membrane potential, and the decreases in reactive oxygen species (ROS) levels and apoptosis rates. Furthermore, Miro1, a critical protein that regulates mitochondrial movement, was knocked down in BMSCs and significantly reduced mitochondrial transfer from BMSCs to NPCs. These results suggested that Miro1 depletion inhibited the rescue of NPCs with mitochondrial dysfunction. Taken together, our data shed light on a novel mechanism by which BMSCs rescue impaired NPCs, providing a concrete foundation to study the critical role of intercellular interactions in disc regeneration., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Fan Yang et al.)

Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.

Stem cells from human exfoliated deciduous teeth (SHED) have stromal-derived inducing activity (SDIA): which means these stromal cells induce neural differentiation where they are used as a substratum for embryonic stem cell (ESCs) culture. Recent studies show that mitochondria or mitochondrial products, as paracrine factors, can be released and transferred from one cell to another. With this information, we were curious to know whether in the SDIA co-culture system, SHED release or donate their mitochondria to ESCs. For this purpose, before co-culture, SHED s' mitochondria and ESCs s' cell membranes were separately labeled with specific fluorescent probes. After co-culture, SHED s' mitochondria were tracked by fluorescent microscope and flow cytometry analysis. Co-culture also performed in the presence of inhibitors that block probable transfer pathways suchlike tunneling nanotubes, gap junctions or vesicles. Results showed that mitochondrial transfer takes place from SHED to ESCs. This transfer partly occurs by tunneling nanotubes and not through gap junctions or vesicles; also was not dependent on intracellular calcium level. This kind of horizontal gene transfer may open a new prospect for further research on probable role of mitochondria on fate choice and neural induction processes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.)

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance., Competing Interests: Declaration of interests Michael T. Heneka serves as an advisory board member at IFM Therapeutics, Alector and Tiaki. He received honoraria for oral presentations from Novartis, Roche, and Biogen. The other authors declare that there is no conflict of interest with regard to the experimental part of this study., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Diversiform ways of intercellular communication are vital links in maintaining homeostasis and disseminating physiological states. Among intercellular bridges, tunneling nanotubes (TNTs) discovered in 2004 were recognized as potential pharmacology targets related to the pathogenesis of common or infrequent neurodegenerative disorders. The neurotoxic aggregates in neurodegenerative diseases including scrapie prion protein (PrPSc), mutant tau protein, amyloid-beta (Aβ) protein, alpha-synuclein (α-syn) as well as mutant Huntington (mHTT) protein could promote TNT formation via certain physiological mechanisms, in turn, mediating the intercellular transmission of neurotoxicity. In this review, we described in detail the skeleton, the formation, the physicochemical properties, and the functions of TNTs, while paying particular attention to the key role of TNTs in the transport of pathological proteins during neurodegeneration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of senescent cells with increasing age is thought to contribute adversely to the development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell-autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of proinflammatory cytokines, chemokines, and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges-or tunnelling nanotubes (TNTs)-containing the cytoskeletal components actin and tubulin, which form direct physical connections between cells. We observe the presence of mitochondria in these TNTs and show organelle transfer through the TNTs to adjacent cells. While transport of individual mitochondria along single TNTs appears by time-lapse studies to be unidirectional, we show by differentially labelled co-culture experiments that organelle transfer through TNTs can occur between different cells of equivalent cell age, but that senescent cells, rather than proliferating cells, appear to be predominant mitochondrial donors. Using small molecule inhibitors, we demonstrate that senescent cell TNTs are dependent on signalling through the mTOR pathway, which we further show is mediated at least in part through the downstream actin-cytoskeleton regulatory factor CDC42. These findings have significant implications for the development of senomodifying therapies, as they highlight the need to account for local direct cell-cell contacts as well as the SASP in order to treat cancer and diseases of ageing in which senescence is a key factor., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Hannah E. Walters and Lynne S. Cox.)

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.

It was already suggested in the early '70's that RNA molecules might transfer between mammalian cells in culture. Yet, more direct evidence for RNA transfer in animal and plant cells was only provided decades later, as this field became established. In this mini-review, we will describe evidence for the transfer of different types of RNA between cells through tunneling nanotubes (TNTs). TNTs are long, yet thin, open-ended cellular protrusions that are structurally distinct from filopodia. TNTs connect cells and can transfer many types of cargo, including small molecules, proteins, vesicles, pathogens, and organelles. Recent work has shown that TNTs can also transfer mRNAs, viral RNAs and non-coding RNAs. Here, we will review the evidence for TNT-mediated RNA transfer, discuss the technical challenges in this field, and conjecture about the possible significance of this pathway in health and disease., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest regarding the publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.)

Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles., Competing Interests: CYL is an inventor on US patents #6,077,938 (Title: Monoclonal antibody to an 80-kDa protease) and #6,677,377 (Title: Structure based discovery of inhibitors of matriptase for the cancer diagnosis and therapy by detection and inhibition of matriptase activity) and MDJ and CYL are inventors on US patent #7,355,015 (Title: Matriptase, a serine protease and its applications). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

F-actin binding and bundling are crucial to a plethora of cell processes, including morphogenesis, migration, adhesion and many others. SWAP-70 was recently described as an in vitro F-actin-binding and -bundling protein. Fluorescence cross-correlation spectroscopy measurements with purified recombinant SWAP-70 confirmed that it forms stable oligomers that facilitate F-actin bundling. However, it remained unclear how SWAP-70 oligomerization and F-actin binding are controlled in living cells. We addressed this by biophysical approaches, including seFRET, FACS-FRET and FLIM-FRET. PIP 3 -mediated association with the cytoplasmic membrane and non-phosphorylated Y426 are required for SWAP-70 to dimerize and to bind F-actin. The dimerization region was identified near the C terminus where R546 is required for dimerization and, thus, F-actin bundling. The in vitro and in vivo data presented here reveal the functional relationship between the cytoplasm-to-membrane translocation and dimerization of SWAP-70, and F-actin binding and bundling, and demonstrate that SWAP-70 is a finely controlled modulator of membrane-proximal F-actin dynamics.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication. IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses., (Copyright © 2020 American Society for Microbiology.)

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca 2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca 2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin., (© 2019 Federation of American Societies for Experimental Biology.)

Caveolins, major components of small plasma membrane invaginations called caveolae, play a role in signaling, particularly in mechanosignaling. These proteins are known to interact with a variety of effector molecules, including G-protein-coupled receptors, Src family kinases, ion channels, endothelial nitric oxide synthase (eNOS), adenylyl cyclases, protein kinase A (PKA), and mitogen-activated PKs (MAPKs). There is, however, speculation on the relevance of these interactions and the mechanisms by which caveolins may control intracellular signaling. This chapter introduces a method of isolation of giant plasma membrane-derived vesicles (GPMVs), which possess full complexity of membrane they originate from, thus comprising an excellent platform to revisit some of the previously described interactions in a cleaner environment and possibly identifying new binding partners. It is also a powerful technique for studying membrane mechanics, as it was previously used to demonstrate the role of caveolae in mechanoprotection.

Many cellular processes rely on precise and timely deformation of the cell membrane. While many proteins participate in membrane reshaping and scission, usually in highly specialized ways, Bin/amphiphysin/Rvs (BAR) domain proteins play a pervasive role, as they not only participate in many aspects of cell trafficking but also are highly versatile membrane remodelers. Subtle changes in the shape and size of the BAR domain can greatly impact the way in which BAR domain proteins interact with the membrane. Furthermore, the activity of BAR domain proteins can be tuned by external physical parameters, and so they behave differently depending on protein surface density, membrane tension, or membrane shape. These proteins can form 3D structures that mold the membrane and alter its liquid properties, even promoting scission under various circumstances.As such, BAR domain proteins have numerous roles within the cell. Endocytosis is among the most highly studied processes in which BAR domain proteins take on important roles. Over the years, a more complete picture has emerged in which BAR domain proteins are tied to almost all intracellular compartments; examples include endosomal sorting and tubular networks in the endoplasmic reticulum and T-tubules. These proteins also have a role in autophagy, and their activity has been linked with cancer. Here, we briefly review the history of BAR domain protein discovery, discuss the mechanisms by which BAR domain proteins induce curvature, and attempt to settle important controversies in the field. Finally, we review BAR domain proteins in the context of a cell, highlighting their emerging roles in cell signaling and organelle shaping.

Freely crawling cells are often viewed as randomly moving Brownian particles but they generally exhibit some directional persistence. This property is often related to their zigzag motile behaviors that can be described as a noisy but temporally structured sequence of "runs" and "turns." However, its underlying biophysical mechanism is largely unexplored. Here, we carefully investigate the collective actin wave dynamics associated with the zigzag-crawling movements of microglia (as primary brain immune cells) employing a number of different quantitative imaging modalities including synthetic aperture microscopy and optical diffraction tomography, as well as conventional fluorescence imaging and scanning electron microscopy. Interestingly, we find that microglia exhibit two distinct types of actin waves working at two quite different time scales and locations, and they seem to serve different purposes. One type of actin waves is fast "peripheral ruffles" arising spontaneously with an oscillating period of about 6 seconds at some portion of the leading edge of crawling microglia, where the vigorously biased peripheral ruffles seem to set the direction of a new turn (in 2-D free space). When the cell turning events are inhibited with a physical confinement (in 1-D track), the peripheral ruffles still exist at the leading edge with no bias but showing phase coherence in the cell crawling direction. The other type is "dorsal actin waves" which also exhibits an oscillatory behavior but with a much longer period of around 2 minutes compared to the fast "peripheral ruffles". Dorsal actin waves (whether the cell turning events are inhibited or not) initiate in the lamellipodium just behind the leading edge, travelling down toward the core region of the cell and disappear. Such dorsal wave propagations seem to be correlated with migration of the cell. Thus, we may view the dorsal actin waves are connected with the "run" stage of cell body, whereas the fast ruffles at the leading front are involved in the "turn" stage., Competing Interests: The authors have declared that no competing interests exist.

Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.

Mutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE's precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc1-GFP and Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which is required for both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie's role in sensory hair cells is to target and stabilize Tmc channel subunits to the site of MET., Competing Interests: The authors have declared that no competing interests exist.

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.)

Glycosphingolipids (GSLs) exhibit a variety of functions in cellular differentiation and interaction. Also, they are known to play a role as receptors in pathogen invasion. A less well-explored feature is the role of GSLs in immune cell function which is the subject of this review article. Here we summarize knowledge on GSL expression patterns in different immune cells. We review the changes in GSL expression during immune cell development and differentiation, maturation, and activation. Furthermore, we review how immune cell GSLs impact membrane organization, molecular signaling, and trans-interactions in cellular cross-talk. Another aspect covered is the role of GSLs as targets of antibody-based immunity in cancer. We expect that recent advances in analytical and genome editing technologies will help in the coming years to further our knowledge on the role of GSLs as modulators of immune cell function.

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses., (© 2018 Condon et al.)

Membrane blebbing-dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D-environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D-collagen, while RhoB overexpression enhanced blebbing and 3D-collagen migration in a manner dependent on its plasma membrane localization and down-stream effectors ROCK and Myosin II RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11-positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D-migration of ALL cells. In conclusion, KIF13A-mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Nanoplasmonic sensors have emerged as a promising measurement approach to track biomacromolecular interactions involving lipid membrane interfaces. By taking advantage of nanoscale fabrication capabilities, it is possible to design sensing platforms with various architectural configurations. Such capabilities open the door to fabricating lipid membrane-coated nanoplasmonic sensors with varying degrees of membrane curvature in order to understand how biomacromolecular interaction processes are influenced by membrane curvature. Herein, we employed an indirect nanoplasmonic sensing approach to characterize the fabrication of supported lipid bilayers (SLBs) on silica-coated nanowell and nanodisk sensing platforms and to investigate how membrane curvature influences membrane-peptide interactions by evaluating the corresponding measurement responses from different spectral signatures that are sensitive to specific regions of the sensor geometries. SLBs were prepared by the vesicle fusion method, as monitored in real-time by nanoplasmonic sensing measurements and further characterized by fluorescence recovery after photobleaching (FRAP) experiments. By resolving different spectral signatures in the nanoplasmonic sensing measurements, it was determined that peptide binding induces membrane disruption at positively curved membrane regions, while peptide binding without subsequent disruption was observed at planar and negatively curved regions. These findings are consistent with the peptide's known preference to selectively form pores in positively curved membranes, providing validation to the nanoplasmonic sensing approach and highlighting how the integration of nanoplasmonic sensors with different nanoscale architectures can be utilized to study the influence of membrane curvature on biomacromolecular interaction processes.

The formation of neurites is an important process affecting the cognitive abilities of an organism. Neurite growth requires the addition of new membranes, but the metabolic remodeling necessary to supply lipids for membrane expansion is poorly understood. Here, we show that synaptic activity, one of the most important inducers of neurite growth, transcriptionally regulates the expression of neuronal glucose transporter Glut3 and rate-limiting enzymes of glycolysis, resulting in enhanced glucose uptake and metabolism that is partly used for lipid synthesis. Mechanistically, CREB regulates the expression of Glut3 and Siah2, the latter and LDH activity promoting the normoxic stabilization of HIF-1α that regulates the expression of rate-limiting genes of glycolysis. The expression of dominant-negative HIF-1α or Glut3 knockdown blocks activity-dependent neurite growth in vitro while pharmacological inhibition of the glycolysis and specific ablation of HIF-1α in early postnatal mice impairs the neurite architecture. These results suggest that the manipulation of neuronal glucose metabolism could be used to treat some brain developmental disorders., (© 2018 The Authors.)

Moseley discusses the molecular and mechanical functions of eisosomes - invaginations from the yeast plasma membrane., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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).)

The amyloid hypothesis of Alzheimer's disease suggests that synaptic degeneration and pathology is caused by the accumulation of amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP). Subsequently, soluble Aβ oligomers cause the loss of synaptic proteins from neurons, a histopathological feature of Alzheimer's disease that correlates with the degree of dementia. In this study, the production of toxic forms of Aβ was examined in vitro using 7PA2 cells stably transfected with human APP. We show that conditioned media from 7PA2 cells containing Aβ oligomers caused synapse degeneration as measured by the loss of synaptic proteins, including synaptophysin and cysteine-string protein, from cultured neurons. Critically, conditioned media from 7PA2 cells treated with valproic acid (2-propylpentanoic acid (VPA)) or propylisopropylacetic acid (PIA) did not cause synapse damage. Treatment with VPA or PIA did not significantly affect total Aβ 42 concentrations; rather these drugs selectively reduced the concentrations of Aβ 42 oligomers in conditioned media. In contrast, treatment significantly increased the concentrations of Aβ 42 monomers in conditioned media. VPA or PIA treatment reduced the concentrations of APP within lipid rafts, membrane compartments associated with Aβ production. These effects of VPA and PIA were reversed by the addition of platelet-activating factor, a bioactive phospholipid produced following activation of phospholipase A 2 , an enzyme sensitive to VPA and PIA. Collectively these data suggest that VPA and PIA reduce Aβ oligomers through inhibition of phospholipase A 2 and suggest a novel therapeutic approach to Alzheimer's treatment., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model., (© 2017 The Author(s).)

Cholesterol is a key component of the cell plasma membrane. It has been suggested that the t-tubule membrane of cardiac ventricular myocytes is enriched in cholesterol and that this plays a role in determining t-tubule structure and function. We have used methyl- β -cyclodextrin (M β CD) to deplete cholesterol in intact and detubulated mouse ventricular myocytes to investigate the contribution of cholesterol to t-tubule structure, membrane capacitance, and the distribution of Ca flux pathways. Depletion of membrane cholesterol was confirmed using filipin; however, di-8-ANEPPS staining showed no differences in t-tubule structure following M β CD treatment. M β CD treatment had no significant effect on the capacitance:volume relationship of intact myocytes or on the decrease in capacitance:volume caused by detubulation. Similarly, Ca influx and efflux were not altered by M β CD treatment and were reduced by a similar amount following detubulation in untreated and M β CD-treated cells. These data show that cholesterol depletion has similar effects on the surface and t-tubule membranes and suggest that cholesterol plays no acute role in determining t-tubule structure and function., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral-but not the apical-plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion., (© 2017 Giovannone, Reales, 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).)

Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF-SRF (myocardin-related transcription factor-serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis., (© 2017 Soto Hinojosa et al.)

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation., (© 2017 Valdivia 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).)

The need for efficient and cost-effective cholera vaccine hasn't lost its actuality in view of the emergence of new strains leading to severe clinical forms of cholera and capable to replace strains of the seventh.cholera pandemic, and in connection with the threat of cholera spreading beyond the borders of endemic countries. In this review data from literature sources are presented about the use of outer membrane proteins, vesicles, cell ghosts of the cholera causative agent in specific prophylaxis and diagnostics of the disease.

The bacterial flagellum is a motility organelle. The flagellum is composed of three main structures: the basal body as a rotary engine embedded in the cellular membranes and cell wall, the long external filament that acts as a propeller, and the hook acting as a universal joint that connects them. I describe protocols for the purification of the filament and hook-basal body from Salmonella enterica serovar Typhimurium.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastases. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in metastatic cell lines treated with TGFβ. Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage., Competing Interests: the authors declare no competing financial interest.