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Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.

SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a significant decrease in the rate of leading edge actin extension in WAVE2 KO cells, and an increase in WAVE1 KO cells. The faster rates of actin extension in WAVE1 KO cells are offset by faster retrograde flow, and therefore do not translate into faster lamellipodium protrusion. Thus, WAVE1 restricts the rate of actin extension at the leading edge, and appears to couple actin networks to the membrane to drive protrusion. Overall, these results suggest that WAVE1 and WAVE2 have redundant roles in promoting Arp2/3-dependent actin nucleation and lamellipodia formation, but distinct roles in controlling actin network extension and harnessing network growth to cell protrusion.

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions arranged in elaborate maze-like patterns on the surface of mucosal epithelial cells. We found that microridges on zebrafish skin cells contained both actin and keratin filaments. Keratin filaments stabilized microridges, and overexpressing keratins lengthened them. Envoplakin and periplakin, plakin family cytolinkers that bind F-actin and keratins, localized to microridges, and were required for their morphogenesis. Strikingly, plakin protein levels directly dictate microridge length. An actin-binding domain of periplakin was required to initiate microridge morphogenesis, whereas periplakin-keratin binding was required to elongate microridges. These findings separate microridge morphogenesis into distinct steps, expand our understanding of intermediate filament functions, and identify microridges as protrusions that integrate actin and intermediate filaments., Competing Interests: YI, VC, Av, LC, AS No competing interests declared, (© 2020, Inaba et al.)

Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering an abnormal accumulation of membrane structures inside the cells (or membrane proliferation) share two major common features: (1) they promote the formation of highly curved membrane domains and (2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, and inspire new applications, either for academia (high-yield membrane protein production and nanovesicle production) or industry (biofuel production and vaccine preparation).

Development of metastasis causes the most serious clinical consequences of cancer and is responsible for over 90 % of cancer-related deaths. Hence, a better understanding of the mechanisms that drive metastasis formation appears critical for drug development designed to prevent the spread of cancer and related mortality. Metastasis dissemination is a multistep process supported by the increased motility and invasiveness capacities of tumor cells. To succeed in overcoming the mechanical constraints imposed by the basement membrane and surrounding tissues, cancer cells reorganize their focal adhesions or extend acto-adhesive cellular protrusions, called invadosomes, that can both contact the extracellular matrix and tune its degradation through metalloprotease activity. Over the last decade, accumulating evidence has demonstrated that altered Ca 2+ channel activities and/or expression promote tumor cell-specific phenotypic changes, such as exacerbated migration and invasion capacities, leading to metastasis formation. While several studies have addressed the molecular basis of Ca 2+ channel-dependent cancer cell migration, we are still far from having a comprehensive vision of the Ca 2+ channel-regulated mechanisms of migration/invasion. This is especially true regarding the specific context of invadosome-driven invasion. This review aims to provide an overview of the current evidence supporting a central role for Ca 2+ channel-dependent signaling in the regulation of these dynamic degradative structures. It will present available data on the few Ca 2+ channels that have been studied in that specific context and discuss some potential interesting actors that have not been fully explored yet., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Actin-microtubule crosstalk is implicated in the formation of cellular protrusions, but the mechanism remains unclear. In this study, we examined the regulation of cell protrusion involving a ubiquitously expressed microtubule-associated protein (MAP) 4, and its superfamily proteins, neuronal MAP2 and tau. Fluorescence microscopy revealed that these MAPs bound to F-actin and microtubules simultaneously, and formed F-actin/microtubule hybrid bundles. The hybrid bundle-forming activity was in the order of MAP2 > MAP4 ≫ tau. Interestingly, the microtubule assembly-promoting activity of MAP4 and MAP2, but not of tau, was upregulated by their interaction with F-actin. When MAP4 was overexpressed in NG108-15 cells, the number of cell processes and maximum process length of each cell increased significantly by 28% and 30%, respectively. Super-resolution microscopy revealed that 95% of microtubules in cell processes colocalized with F-actin, and MAP4 was always found in their vicinity. These results suggest that microtubule elongation along F-actin induced by MAP4 contributes to the formation of cellular protrusions. Since MAP4, MAP2 and tau had different crosstalk activity between F-actin and microtubules, it is likely that the functional differentiation of these MAPs is a driving force for neural evolution, causing significant changes in cell morphology., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them., Methods: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays., Results: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly., Conclusions: Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells. Video abstract.

Tractional remodeling of collagen fibrils by fibroblasts requires long cell extensions that mediate fibril alignment. The formation of these cell extensions involves flightless I (FliI), an actin-binding protein that contains a leucine-rich-repeat (LRR), which binds R-ras and may regulate cdc42. We considered that FliI interacts with small GTPases and their regulators to mediate assembly of cell extensions. Mass spectrometry analyses of FliI immunoprecipitates showed abundant Ras GTPase-activating-like protein (IQGAP1), which in immunostained samples colocalized with FliI at cell adhesions. Knockdown of IQGAP1 reduced the numbers of cell extensions and the alignment of collagen fibrils. In experiments using dominant negative mutants, cdc42 activity was required for the formation of short extensions while R-ras was required for the formation of long extensions. Immunoprecipitation of wild-type and mutant constructs showed that IQGAP1 associated with cdc42 and R-ras; this association required the GAP-related domain (1004-1237 aa) of IQGAP1. In cells transfected with FliI mutants, the LRR of FliI, but not its gelsolin-like domains, mediated association with cdc42, R-ras, and IQGAP1. We conclude that FliI interacts with IQGAP1 and co-ordinates with cdc42 and R-ras to control the formation of cell extensions that enable collagen tractional remodeling.

Microtubule-depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule-depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046-induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock-down expression of FAK inhibited IMB5046-induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF-H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046-induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule-depolymerizing agents to be used as vascular disrupting agents., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

Blood vessels (BVs) are considered an integral component of neural stem cells (NSCs) niches. NSCs in the dentate gyrus (DG(have enigmatic elaborated apical cellular processes that are associated with BVs. Whether this contact serves as a mechanism for delivering circulating molecules is not known. Here we uncovered a previously unrecognized communication route allowing exclusive direct access of blood-borne substances to hippocampal NSCs. BBB-impermeable fluorescent tracer injected transcardially to mice is selectively uptaken by DG NSCs within a minute, via the vessel-associated apical processes. These processes, measured >30 nm in diameter, establish direct membrane-to-membrane contact with endothelial cells in specialized areas of irregular endothelial basement membrane and enriched with vesicular activity. Doxorubicin, a brain-impermeable chemotherapeutic agent, is also readily and selectively uptaken by NSCs and reduces their proliferation, which might explain its problematic anti-neurogenic or cognitive side-effect. The newly-discovered NSC-BV communication route explains how circulatory neurogenic mediators are 'sensed' by NSCs., Competing Interests: TL, ES, BB, MG, SK, TK, AB, EK No competing interests declared, (© 2020, Licht et al.)

Actin-based tubular connections between cells have been observed in many cell types. Termed "tunneling nanotubes (TNTs)," "membrane nanotubes," "tumor microtubes (TMTs)," or "cytonemes," these protrusions interconnect cells in dynamic networks. Structural features in these protrusions vary between cellular systems, including tubule diameter and the presence of microtubules. We find tubular protrusions, which we classify as TMTs, in a pancreatic cancer cell line, Dartmouth-Hitchcock Pancreatic Cancer (DHPC)-018. TMTs are present in DHPC-018-derived tumors in mice, as well as in a mouse model of pancreatic cancer and a subset of primary human tumors. DHPC-018 TMTs have heterogeneous diameter (0.39-5.85 µm, median 1.92 µm) and contain actin filaments, microtubules, and cytokeratin 19-based intermediate filaments. TMTs do not allow intercellular transfer of cytoplasmic GFP. Actin filaments are cortical within the protrusion, as opposed to TNTs, in which filaments run down the center. TMTs are dynamic in length, but are long lived (median >60 min). Inhibition of actin polymerization, but not microtubules, results in TMT loss. Extracellular calcium is necessary for TMT maintenance. A second class of tubular protrusion, which we term cell-substrate protrusion, has similar width range and cytoskeletal features but makes contact with the substratum as opposed to another cell. Similar to previous work on TNTs, we find two assembly mechanisms for TMTs, which we term "pull-away" and "search-and-capture." Inhibition of Arp2/3 complex inhibits TMT assembly by both mechanisms. This work demonstrates that the actin architecture of TMTs in pancreatic cancer cells is fundamentally different from that of TNTs and demonstrates the role of Arp2/3 complex in TMT assembly.

Bacteria can produce membranous nanotubes that mediate contact-dependent exchange of molecules among bacterial cells. However, it is unclear how nanotubes cross the cell wall to emerge from the donor or to penetrate into the recipient cell. Here, we report that Bacillus subtilis utilizes cell wall remodeling enzymes, the LytC amidase and its enhancer LytB, for efficient nanotube extrusion and penetration. Nanotube production is reduced in a lytBC mutant, and the few nanotubes formed appear deficient in penetrating into target cells. Donor-derived LytB molecules localize along nanotubes and on the surface of nanotube-connected neighbouring cells, primarily at sites of nanotube penetration. Furthermore, LytB from donor B. subtilis can activate LytC of recipient bacteria from diverse species, facilitating cell wall hydrolysis to establish nanotube connection. Our data provide a mechanistic view of how intercellular connecting devices can be formed among neighbouring bacteria.

Apoptotic cells form membrane blebs, but little is known about how the formation and dynamics of membrane blebs are regulated. The size of blebs gradually increases during the progression of apoptosis, eventually forming large extracellular vesicles called apoptotic bodies that have immune-modulating activities. In this study, we investigated the molecular mechanism involved in the differentiation of blebs into apoptotic blebs by comparing the dynamics of the bleb formed during cell migration and the bleb formed during apoptosis. We revealed that the enhanced activity of ROCK1 is required for the formation of small blebs in the early phase of apoptosis, which leads to the physical disruption of nuclear membrane and the degradation of Lamin A. In the late phase of apoptosis, the loss of asymmetry in phospholipids distribution caused the enlargement of blebs, which enabled translocation of damage-associated molecular patterns to the bleb cytoplasm and maturation of functional apoptotic blebs. Thus, changes in cell membrane dynamics are closely linked to cytoplasmic changes during apoptotic bleb formation.

Hydrogen peroxide (H 2 O 2 ) is a key redox intermediate generated within cells. Existing probes for H 2 O 2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H 2 O 2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H 2 O 2 diffusion from the mitochondrial matrix, to find a functional output of H 2 O 2 gradients in polarized cells, and to prove the existence of H 2 O 2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H 2 O 2 imaging in various biological contexts., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures that are arranged in maze-like patterns on the apical surfaces of zebrafish skin cells. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A nonmuscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex, and inhibiting NMII blocked apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex can pattern 3D cell surfaces., (© 2020 van Loon et al.)

Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC., Competing Interests: DY, FQ, HC, CC, JL, NJ, BG, CS, CK, MO No competing interests declared, MW has equity in, and is an advisor for, D2GOncology, JS Receives research funding from Stemcentrx/Abbvie, Pfizer, and Revolution Medicines and owns stock in Forty Seven Inc, (© 2019, Yang et al.)

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Brain stem cells stop dividing in late Drosophila embryos and begin dividing again in early larvae after feeding induces reactivation. Quiescent neural stem cells (qNSCs) display an unusual cytoplasmic protrusion that is no longer present in reactivated NSCs. The protrusions join the qNSCs to the neuropil, brain regions that are thought to maintain NSCs in an undifferentiated state, but the function of the protrusions is not known. Here we show that qNSC protrusions contain clustered mitochondria that are likely maintained in position by slow forward-and-backward microtubule growth. Larvae treated with a microtubule-stabilizing drug show bundled microtubules and enhanced mitochondrial clustering in NSCs, together with reduced qNSC reactivation. We further show that intestinal stem cells contain mitochondria-enriched protrusions. The qNSC and intestinal stem-cell protrusions differ from previously reported cytoplasmic extensions by forming stem-cell-to-niche mitochondrial bridges that could potentially both silence genes and sense signals from the stem cell niche., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing evidence during the past two decades shows that cells interconnect and communicate through cytonemes. These cytoskeleton-driven extensions of specialized membrane territories are involved in cell-cell signaling in development, patterning, and differentiation, but also in the maintenance of adult tissue homeostasis, tissue regeneration, and cancer. Brain tumor cells in glioblastoma extend ultralong membrane protrusions (named tumor microtubes, TMs), which contribute to invasion, proliferation, radioresistance, and tumor progression. Here we review the mechanisms underlying cytoneme formation, regulation, and their roles in cell signaling and communication in epithelial cells and other cell types. Furthermore, we discuss the recent discovery of glial cytonemes in the Drosophila glial cells that alter Wingless (Wg)/Frizzled (Fz) signaling between glia and neurons. Research on cytoneme formation, maintenance, and cell signaling mechanisms will help to better understand not only physiological developmental processes and tissue homeostasis but also cancer progression., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this article.

Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.

The eukaryotic pathway of galactolipid synthesis involves fatty acid synthesis in the chloroplast, followed by assembly of phosphatidylcholine (PC) in the endoplasmic reticulum (ER), and then turnover of PC to provide a substrate for chloroplast galactolipid synthesis. However, the mechanisms and classes of lipids transported between the chloroplast and the ER are unclear. PC, PC-derived diacylglycerol, phosphatidic acid, and lyso-phosphatidylcholine (LPC) have all been implicated in ER-to-chloroplast lipid transfer. LPC transport requires lysophosphatidylcholine acyltransferase (LPCAT) activity at the chloroplast to form PC before conversion to galactolipids. However, LPCAT has also been implicated in the opposite chloroplast-to-ER trafficking of newly synthesized fatty acids through PC acyl editing. To understand the role of LPC and LPCAT in acyl trafficking we produced and analyzed the Arabidopsis ( Arabidopsis thaliana ) act1 lpcat1 lpcat2 triple mutant. LPCAT1 and LPCAT2 encode the major lysophospholipid acyltransferase activity of the chloroplast, and it is predominantly for incorporation of nascent fatty acids exported form the chloroplast into PC by acyl editing. In vivo acyl flux analysis revealed eukaryotic galactolipid synthesis is not impaired in act1 lpcat1 lpcat2 and uses a PC pool distinct from that of PC acyl editing. We present a model for the eukaryotic pathway with metabolically distinct pools of PC, suggesting an underlying spatial organization of PC metabolism as part of the ER-chloroplast metabolic interactions., (© 2019 American Society of Plant Biologists. All rights reserved.)

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure-mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area-based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.

GLT-1, the major glutamate transporter, is expressed at perisynaptic astrocytic processes (PAP) and axon terminals (AxT). GLT-1 is coupled to Na+/K+-ATPase (NKA) α1-3 isoforms, whose subcellular distribution and spatial organization in relationship to GLT-1 are largely unknown. Using several microscopy techniques, we showed that at excitatory synapses α1 and α3 are exclusively neuronal (mainly in dendrites and in some AxT), while α2 is predominantly astrocytic. GLT-1 displayed a differential colocalization with α1-3. GLT-1/α2 and GLT-1/α3 colocalization was higher in GLT-1 positive puncta partially (for GLT-1/α2) or almost totally (for GLT-1/α3) overlapping with VGLUT1 positive terminals than in nonoverlapping ones. GLT-1 colocalized with α2 at PAP, and with α1 and α3 at AxT. GLT-1 and α2 gold particles were ∼1.5-2 times closer than GLT-1/α1 and GLT-1/α3 particles. GLT-1/α2 complexes (edge to edge interdistance of gold particles ≤50 nm) concentrated at the perisynaptic region of PAP membranes, whereas neuronal GLT-1/α1 and GLT-1/α3 complexes were fewer and more uniformly distributed in AxT. These data unveil different composition of GLT-1 and α subunits complexes in the glial and neuronal domains of excitatory synapses. The spatial organization of GLT-1/α1-3 complexes suggests that GLT-1/NKA interaction is more efficient in astrocytes than in neurons, further supporting the dominant role of astrocytic GLT-1 in glutamate homeostasis., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Localization of RNAs to various subcellular destinations is a widely used mechanism that regulates a large proportion of transcripts in polarized cells. In many cases, such localized transcripts mediate spatial control of gene expression by being translationally silent while in transit and locally activated at their destination. Here, we investigate the translation of RNAs localized at dynamic cellular protrusions of human and mouse, migrating, mesenchymal cells. In contrast to the model described above, we find that protrusion-localized RNAs are not locally activated solely at protrusions, but can be translated with similar efficiency in both internal and peripheral locations. Interestingly, protrusion-localized RNAs are translated at extending protrusions, they become translationally silenced in retracting protrusions and this silencing is accompanied by coalescence of single RNAs into larger heterogeneous RNA clusters. This work describes a distinct mode of translational regulation of localized RNAs, which we propose is used to regulate protein activities during dynamic cellular responses., Competing Interests: KM, KY, TW, GC, SM No competing interests declared

Tunneling nanotubes (TNTs) are thin membranous tubes that interconnect cells, representing a novel route of cell-to-cell communication and spreading of pathogens. TNTs form between many cell types, yet their inception mechanisms remain elusive. We review in this study general concepts related to the formation and stability of membranous tubular structures with a focus on a deviatoric elasticity model of membrane nanodomains. We review experimental evidence that tubular structures initiate from local membrane bending facilitated by laterally distributed proteins or anisotropic membrane nanodomains. We further discuss the numerical results of several theoretical and simulation models of nanodomain segregation suggesting the mechanisms of TNT inception and stability. We discuss the coupling of nanodomain segregation with the action of protruding cytoskeletal forces, which are mostly provided in eukaryotic cells by the polymerization of f-actin, and review recent inception mechanisms of TNTs in relation to motor proteins., Competing Interests: The authors declare no conflict of interest.

The F-BAR family of proteins play important roles in many cellular processes by regulating both membrane and actin dynamics. The CIP4 family of F-BAR proteins is widely recognized to function in endocytosis by elongating endocytosing vesicles. However, in primary cortical neurons, CIP4 concentrates at the tips of extending lamellipodia and filopodia and inhibits neurite outgrowth. Here, we report that the highly homologous CIP4 family member, FBP17, induces tubular structures in primary cortical neurons and results in precocious neurite formation. Through domain swapping and deletion experiments, we demonstrate that a novel polybasic region between the F-BAR and HR1 domains is required for membrane bending. Moreover, the presence of a poly-PxxP region in longer splice isoforms of CIP4 and FBP17 largely reverses the localization and function of these proteins. Thus, CIP4 and FBP17 function as an antagonistic pair to fine-tune membrane protrusion, endocytosis, and neurite formation during early neuronal development., (© 2019 Taylor et al.)

Tunneling nanotubes (TNT) are thin, membranous, tunnel-like cell-to-cell connections, but the mechanisms underlying their biogenesis or functional role remains obscure. Here, we report, Rhes, a brain-enriched GTPase/SUMO E3-like protein, induces the biogenesis of TNT-like cellular protrusions, "Rhes tunnels," through which Rhes moves from cell to cell and transports Huntington disease (HD) protein, the poly-Q expanded mutant Huntingtin (mHTT). The formation of TNT-like Rhes tunnels requires the Rhes's serine 33, C-terminal CAAX, and a SUMO E3-like domain. Electron microscopy analysis revealed that TNT-like Rhes tunnels appear continuous, cell-cell connections, and <200 nm in diameter. Live-cell imaging shows that Rhes tunnels establish contact with the neighboring cell and deliver Rhes-positive cargoes, which travel across the plasma membrane of the neighboring cell before entering it. The Rhes tunnels carry Rab5a/Lyso 20-positive vesicles and transport mHTT, but not normal HTT, mTOR, or wtTau proteins. SUMOylation-defective mHTT, Rhes C263S (cannot SUMOylate mHTT), or CRISPR/Cas9-mediated depletion of three isoforms of SUMO diminishes Rhes-mediated mHTT transport. Thus, Rhes promotes the biogenesis of TNT-like cellular protrusions and facilitates the cell-cell transport of mHTT involving SUMO-mediated mechanisms., (© 2019 Sharma and Subramaniam.)

Microorganisms require a motility structure to move towards optimal growth conditions. The motility structure from archaea, the archaellum, is fundamentally different from its bacterial counterpart, the flagellum, and is assembled in a similar fashion as type IV pili. The archaellum filament consists of thousands of copies of N-terminally processed archaellin proteins. Several archaea, such as the euryarchaeon Haloarcula marismortui, encode multiple archaellins. Two archaellins of H. marismortui display differential stability under various ionic strengths. This suggests that these proteins behave as ecoparalogs and perform the same function under different environmental conditions. Here, we explored this intriguing system to study the differential regulation of these ecoparalogous archaellins by monitoring their transcription, translation, and assembly into filaments. The salt concentration of the growth medium induced differential expression of the two archaellins. In addition, this analysis indicated that archaellation in H. marismortui is majorly regulated on the level of secretion, by a still unknown mechanism. These findings indicate that in archaea, multiple encoded archaellins are not completely redundant, but in fact can display subtle functional differences, which enable cells to cope with varying environmental conditions., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.

Cell⁻cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their specific functions have not been elucidated due to the lack of a method for their specific isolation and analysis. In this paper, we described, for the first time, a method to specifically isolate distinct protrusion subtypes, based on their morphological structures or fluorescent markers, using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrate that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Our method confirmed that different types of protrusions have distinct proteomes and it promises to advance the characterization and the understanding of these unique structures to shed light on their possible role in health and disease.

Background: The paradigm shift localizing the origin of ovarian high-grade serous carcinoma (HGSC) to the fallopian tube underscores the rationale for meticulous microscopic examination of salpingectomy specimens. The precursor, termed "serous tubal intraepithelial carcinoma," is often a focal lesion, which poses difficulties for histologic diagnosis., Methods: The authors describe a method to examine exfoliated epithelial cells from fallopian tube fimbria by gentle brushing, thereby enabling thorough sampling of the mucosal surface. Fimbrial brushings were collected from 20 fresh salpingectomy specimens from 15 patients, including 5 who had pathologically confirmed ovarian HGSC. Samples taken only from tubes that were grossly negative for tumor were processed for Papanicolaou staining, p53 immunocytochemistry, and tumor protein 53 (TP53) mutation analysis., Results: Cells with malignant cytomorphologic features were identified only in tubal brushings from patients with ovarian HGSC. In all cases, atypical/malignant cells on cytology corresponded to lesions with similar morphology and immunostaining pattern in permanent sections, demonstrating the sensitivity of the technique while providing reassurance that specimen integrity was not disrupted by the procedure. Targeted next-generation sequencing confirmed the presence of TP53 mutations in fimbrial brushings from HGSC, but not in benign samples, and demonstrated concordance with the immunostaining pattern. Identical mutations were observed in matched lesions microdissected from formalin-fixed tissue sections., Conclusions: The described technique enables cytologic evaluation of the fallopian tube fimbria for a diagnosis of serous tubal intraepithelial carcinoma, serving as a complement to histology while offering distinct advantages with respect to the procurement of cellular material for ancillary testing and research., (© 2019 American Cancer Society.)

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1 + cells) regulate immune responses 1-5 . CX3CR1 + cells take up luminal antigens by protruding their dendrites into the lumen 1-4,6 . However, it remains unclear how dendrite protrusion by CX3CR1 + cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1 + cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1 + cells, showed defective dendrite protrusions of CX3CR1 + cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1 + cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1 + cells of wild-type mice, but not of Gpr31b -/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1 + cells in the small intestine of wild-type mice, but not in that of Gpr31b -/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1 + cells.

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral "lamellar smooth muscle cells" (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing β-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in "dense bodies," "focal adhesions," and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions.

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber-mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies., (© 2018 Wiley Periodicals, Inc.)

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined., Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading., Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells., Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.

Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age-dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Programmed cell death (PCD) is a conserved phenomenon in multicellular organisms required to maintain homeostasis. Among the regulated cell death pathways, apoptosis is a well-described form of PCD in mammalian cells. One of the characteristic features of apoptosis is the change in cellular morphology, often leading to the fragmentation of the cell into smaller membrane-bound vesicles through a process called apoptotic cell disassembly. Interestingly, some of these morphological changes and cell disassembly are also noted in cells of other organisms including plants, fungi and protists while undergoing 'apoptosis-like PCD'. This review will describe morphologic features leading to apoptotic cell disassembly, as well as its regulation and function in mammalian cells. The occurrence of cell disassembly during cell death in other organisms namely zebrafish, fly and worm, as well as in other eukaryotic cells will also be discussed.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During development and in cancer, cells often move together in small to large collectives. To move as a unit, cells within collectives need to stay coupled together and coordinate their motility. How cell collectives remain interconnected and migratory, especially when moving through in vivo environments, is not well understood. The genetically tractable border cell group undergoes a highly polarized and cohesive cluster-type migration in the Drosophila ovary. Here we report that the small GTPase Rap1, through activation by PDZ-GEF, regulates border cell collective migration. We find that Rap1 maintains cell contacts within the cluster, at least in part by promoting the organized distribution of E-cadherin at specific cell-cell junctions. Rap1 also restricts migratory protrusions to the front of the border cell cluster and promotes the extension of protrusions with normal dynamics. Further, Rap1 is required in the outer migratory border cells but not in the central nonmigratory polar cells. Such cell specificity correlates well with the spatial distribution of the inhibitory Rapgap1 protein, which is higher in polar cells than in border cells. We propose that precisely regulated Rap1 activity reinforces connections between cells and polarizes the cluster, thus facilitating the coordinated collective migration of border cells.

Rho guanine nucleotide exchange factor 4 (ARHGEF4) is a guanine nucleotide exchange factor that is specific for Rac1 and Cdc42. The aim of the present study was to investigate the role of ARHGEF4 in the motility and invasiveness of pancreatic cancer cells. Evaluation of an immunohistochemical staining of 102 resected pancreatic cancer samples demonstrated that high ARHGEF4 expression was correlated with an independent predictor of worse overall survival in univariate and multivariate analyses. Immunofluorescence analyses and Matrigel invasion assays demonstrated that suppression of ARHGEF4 inhibited the formation of membrane protrusions, and in turn inhibited cell motility and invasion. A phosphoprotein array analysis demonstrated that knockdown of ARHGEF4 decreased phosphorylated extracellular signal-regulated kinase (ERK)1/2 and glycogen synthase kinase-3 (GSK-3)α/β in pancreatic cancer cells, and ERK1/2 and GSK-3α/β were associated with ARHGEF4-related motility and invasiveness through an increase in cell protrusions. These results suggested that ARHGEF4 stimulates ERK1/2 and GSK-3α/β, and provided evidence that ARHGEF4 promotes cell motility and invasiveness. Inhibition of ARHGEF4 may be a novel approach to a targeted molecular therapy, as any such therapy would limit the motility and invasiveness of pancreatic cancer cells.

Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.