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Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions is unclear. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two nucleation-promoting factors for the Arp2/3 complex, WAVE and N-WASP, exhibit different localization patterns in the protrusions. Whereas WAVE is closely associated with the plasma membrane at the leading edge of the protrusive structures, N-WASP is enriched with WIP along the actin bundles in the shafts of the protrusions. During protrusion initiation and growth, the Arp2/3 complex nucleates branched actin filaments to generate low-density actin clouds in which the large GTPase dynamin organizes the new branched actin filaments into bundles, followed by actin-bundle stabilization by WIP, the latter functioning as an actin-bundling protein. Disruption of any of these components results in defective protrusions and failed myoblast fusion in cultured cells and mouse embryos. Together, our study has revealed the intricate spatiotemporal coordination between two nucleation-promoting factors and two actin-bundling proteins in building invasive protrusions at the mammalian fusogenic synapse and has general implications in understanding invasive protrusion formation in cellular processes beyond cell-cell fusion., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin T. vaginalis TSP5 protein (TvTSP5), which is localized on the cell surface of the parasite, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate that parasites that overexpress TvTSP5 possess an increased ability to adhere to host cells, enhanced aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of T. vaginalis interactions with host cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Membrane ruffles are cell actin-based membrane protrusions that have distinct structural characteristics. Linear ruffles with columnar spike-like and veil-like structures assemble at the leading edge of cell membranes. Circular dorsal ruffles (CDRs) have no supporting columnar structures but their veil-like structures, connecting from end to end, present an enclosed ring-shaped circular outline. Membrane ruffles are involved in multiple cell functions such as cell motility, macropinocytosis, receptor internalization, fluid viscosity sensing in a two-dimensional culture environment, and protecting cells from death in response to physiologically compressive loads. Herein, we review the state-of-the-art knowledge on membrane ruffle structure and function, the growth factor-induced membrane ruffling process, and the growth factor-independent ruffling mode triggered by calcium and other stimulating factors, together with the respective underlying mechanisms. We also summarize the inhibitors used in ruffle formation studies and their specificity. In the last part, an overview is given of the various techniques in which the membrane ruffles have been visualized up to now.

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape., (© 2024 Federation of European Biochemical Societies.)

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by formation of lamellipodia and/or filopodia, while amoeboid migration is based on bleb formation. Changing of migrational conditions can lead to alteration in the character of cell movement. For example, inhibition of the Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility mode. Ability of the cells to switch from one type of motility to another is called migratory plasticity. Cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryo fibroblasts REF52 with normal VIF organization, fibroblasts with vimentin knockout (REF -/- ), and fibroblasts with mutation inhibiting assembly of the full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase in the number of cells with blebs. The fibroblasts with short fragments of vimentin demonstrate the significant increase in number of cells forming blebs both spontaneously and in the presence of CK-666. Disruption of the VIF organization did not lead to the significant changes in the microtubules network or the level of myosin light chain phosphorylation, but caused significant reduction in the focal contact system. The most pronounced and statistically significant decrease in both size and number of focal adhesions were observed in the REF117 cells. We believe that regulation of the membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that organization of VIFs determines the type of cell protrusions, which, in turn, determines the character of cell movement. A novel role of VIFs as a regulator of membrane blebbing, essential for manifestation of the migratory plasticity, is shown.

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins., (Copyright © 2024. Published by Elsevier Inc.)

LncRNAs are emerging as important regulators of gene expression by controlling transcription in the nucleus and by modulating mRNA translation in the cytoplasm. In this study, we reveal a novel function of lncRNA SNHG15 in mediating breast cancer cell invasion through regulating the local translation of CDH2 mRNA. We show that SNHG15 preferentially localizes at the cellular protrusions or cell leading edge and that this localization is directed by IMP1, a multifunctional protein involved in many aspects of RNA regulation. We demonstrate that SNHG15 also forms a complex with nucleolin, allowing nucleolin to be co-transported with SNHG15 to the cell protrusions, where the accumulated nucleolin is able to bind to CDH2 mRNA. Interaction with nucleolin stabilizes local CDH2 mRNA and regulates its translation, thus promoting cell invasive potential. Our findings reveal an underlying mechanism by which lncRNA could serve as a carrier to transport a protein regulator into a specific cell compartment to enhance target mRNA expression.

Adherent cells migrate on layered tissue interfaces to drive morphogenesis, wound healing, and tumor invasion. Although stiffer surfaces are known to enhance cell migration, it remains unclear whether cells sense basal stiff environments buried under softer, fibrous matrix. Using layered collagen-polyacrylamide gel systems, we unveil a migration phenotype driven by cell-matrix polarity. Here, cancer (but not normal) cells with stiff base matrix generate stable protrusions, faster migration, and greater collagen deformation because of "depth mechanosensing" through the top collagen layer. Cancer cell protrusions with front-rear polarity produce polarized collagen stiffening and deformations. Disruption of either extracellular or intracellular polarity via collagen crosslinking, laser ablation, or Arp2/3 inhibition independently abrogates depth-mechanosensitive migration of cancer cells. Our experimental findings, validated by lattice-based energy minimization modeling, present a cell migration mechanism whereby polarized cellular protrusions and contractility are reciprocated by mechanical extracellular polarity, culminating in a cell-type-dependent ability to mechanosense through matrix layers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death 1 . Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue 2,3 . In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs 4-11 . Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios 12 . Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration., (© 2022. The Author(s).)

Cellular protrusions generated by the actin cytoskeleton are central to the process of building the body of the embryo. Problems with cellular protrusions underlie human diseases and syndromes, including implantation defects and pregnancy loss, congenital birth defects, and cancer. Cells use protrusive activity together with actin-myosin contractility to create an ordered body shape of the embryo. Here, I review how actin-rich protrusions are used by two major morphological cell types, epithelial and mesenchymal cells, during collective cell migration to sculpt the mouse embryo body. Pre-gastrulation epithelial collective migration of the anterior visceral endoderm is essential for establishing the anterior-posterior body axis. Gastrulation mesenchymal collective migration of the mesoderm wings is crucial for body elongation, and somite and heart formation. Analysis of mouse mutants with disrupted cellular protrusions revealed the key role of protrusions in embryonic morphogenesis and embryo survival. Recent technical approaches have allowed examination of the mechanisms that control cell and tissue movements in vivo in the complex 3D microenvironment of living mouse embryos. Advancing our understanding of protrusion-driven morphogenesis should provide novel insights into human developmental disorders and cancer metastasis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cell-cell communications are central to a variety of physiological and pathological processes in multicellular organisms. Cells often rely on cellular protrusions to communicate with one another, which enable highly selective and efficient signaling within complex tissues. Owing to significant improvements in imaging techniques, identification of signaling protrusions has increased in recent years. These protrusions are structurally specialized for signaling and facilitate interactions between cells. Therefore, physical regulation of these structures must be key for the appropriate strength and pattern of signaling outcomes. However, the typical approaches for understanding signaling regulation tend to focus solely on changes in signaling molecules, such as gene expression, protein-protein interaction, and degradation. In this short review, we summarize the studies proposing the removal of different types of signaling protrusions-including cilia, neurites, MT (microtubule based)-nanotubes and microvilli-and discuss their mechanisms and significance in signaling regulation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of β1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix., (© 2022 Campisi et al.)

Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery., Competing Interests: The authors have declared that no competing interests exist.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport., (© 2021 Pichon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρ R , and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρ R , as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism., Competing Interests: Declaration of interests The authors declare no competing or financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

There is a strong association between arsenic exposure and lung cancer development, however, the mechanism by which arsenic exposure leads to carcinogenesis is not clear. In our previous study, we observed that when BEAS-2B cells are chronically exposed to arsenic, there is an increase in secreted TGFα, as well as an increase in EGFR expression and activity. Further, these changes were broadly accompanied with an increase in cell migration. The overarching goal of this study was to acquire finer resolution of the arsenic-dependent changes in cell migration, as well as to understand the role of increased EGFR expression and activity levels in the underlying mechanisms of cell migration. To do this, we used a combination of biochemical and single cell assays, and observed chronic arsenic treatment enhancing cell migration by increasing cell speed, cell persistence and cell protrusion length. All three parameters were further increased by the addition of TGFα, indicating EGFR activity is sufficient to enhance those aspects of cell migration. In contrast, EGFR activity was necessary for the increase in cell speed, as it was reversed with an EGFR inhibitor, AG1478, but was not necessary to enhance persistence and protrusion length. From these data, we were able to isolate both EGFR-dependent and -independent features of cell migration that were enhanced by chronic arsenic exposure., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2 -/- mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2 -/- MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein-protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2 -/- MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers., (© 2021 John Wiley & Sons Ltd.)

Radial glial cells (RGC) are at the center of brain development in vertebrates, acting as progenitors for neurons and macroglia (oligodendrocytes and astrocytes) and as guides for migration of neurons from the ventricular surface to their final positions in the brain. These cells originate from neuroepithelial cells (NEC) from which they inherit their epithelial features and polarized morphology, with processes extending from the ventricular to the pial surface of the embryonic cerebrum. We have learnt a great deal since the first descriptions of these cells at the end of the nineteenth century. However, there are still questions regarding how and when NEC transform into RGC or about the function of intermediate filaments such as glial fibrillary acidic protein (GFAP) in RGCs and their dynamics during neurogenesis. For example, it is not clear why RGCs in primates, including humans, express GFAP at the onset of cortical neurogenesis while in rodents it is expressed when it is essentially complete. Based on an ultrastructural analysis of GFAP expression and cell morphology of dividing progenitors in the developing neocortex of the macaque monkey, we show that RGCs become the main progenitor in the developing cerebrum by the start of neurogenesis, as all dividing cells show glial features such as GFAP expression and lack of tight junctions. Also, our data suggest that RGCs retract their apical process during mitosis. We discuss our findings in the context of the role and molecular characteristics of RGCs in the vertebrate brain, their differences with NECs and their dynamic behavior during the process of neurogenesis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Circular dorsal ruffles (CDRs) are a kind of special ring-shaped membrane structure rich in F-actin, it is highly involved in the invasion-metastasis of tumor. Shear stress is one of the biophysical elements that affects the fate of tumor cells. However, how shear stress contributes to the CDRs formation is still unclear. In this study, we found that shear stress stimulated the formation of CDRs and promoted the migration of human breast MDA-MB-231 carcinoma cells. Integrin-linked kinase (ILK) mediated the recruiting of ADP-ribosylation factors (ARAP1/Arf1) to CDRs. Meanwhile, the transfection of ARAP1 or Arf1 mutant decreased the number of cells with CDRs, the CDRs areas and perimeters, thus blocked the cancer cell migration. This indicated that the ARAP1/Arf1 were necessary for the CDRs formation and cancer cell migration. Further study revealed that shear stress could stimulate the formation of intracellular macropinocytosis (MPS) thus promoted the ARAP1/Arf1 transportation to early endosome to regulate cancer cell migration after the depolymerization of CDRs. Our study elucidates that the CDRs formation is essential in shear stress-induced breast cancer cell migration, which provides a new research target for exploring the cytoskeletal mechanisms of breast cancer malignance., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Teruo Fujii reports financial support was provided by the Japanese Agency for Medical Research and Development, Tokyo, Japan. Kotaro Doi, Hiroshi Kimura, Tetsuhiro Tanaka, Reiko Inagi, Masaomi Nangaku reports financial support was provided by the Japanese Agency for Medical Research and Development, Tokyo, Japan., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion., Competing Interests: The authors declare no competing interest.

Melanoma cells have been shown to undergo fast amoeboid (leader bleb-based) migration, requiring a single large bleb for migration. In leader blebs, is a rapid flow of cortical actin that drives the cell forward. Using RNAi, we find that co-depleting cofilin-1 and actin depolymerizing factor (ADF) led to a large increase in cortical actin, suggesting that both proteins regulate cortical actin. Furthermore, severing factors can promote contractility through the regulation of actin architecture. However, RNAi of cofilin-1 but not ADF led to a significant decrease in cell stiffness. We found cofilin-1 to be enriched at leader bleb necks, whereas RNAi of cofilin-1 and ADF reduced bleb sizes and the frequency of motile cells. Strikingly, cells without cofilin-1 and ADF had blebs with abnormally long necks. Many of these blebs failed to retract and displayed slow actin turnover. Collectively, our data identifies cofilin-1 and ADF as actin remodeling factors required for fast amoeboid migration., Competing Interests: MU, JL No competing interests declared, (© 2021, Ullo and Logue.)

Cadherins harness the actin cytoskeleton to build cohesive sheets of cells using paradoxically weak bonds, but the molecular mechanisms are poorly understood. In one popular model, actin organizes cadherins into large, micrometer-sized clusters known as puncta. Myosin is thought to pull on these puncta to generate strong adhesion. Here, however, we show that cadherin puncta are actually interdigitated actin microspikes generated by actin polymerization mediated by three factors (Arp2/3, EVL, and CRMP-1). The convoluted membranes in these regions give the impression of cadherin clustering by fluorescence microscopy, but the ratio of cadherin to membrane is constant. Nevertheless, these interlocking fingers of membrane are important for adhesion because perturbing their formation disrupts cell adhesion. In contrast, blocking myosin-dependent contractility does not disrupt either the interdigitated microspikes or lateral membrane adhesion. "Puncta" are zones of strong cell-cell adhesion not due to cadherin clustering but that occur because the interdigitated microspikes expand the surface area available for adhesive bond formation and increase the asperity of the cell surface to promote friction between cells., Competing Interests: The authors declare no competing interest.

Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long time scales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, efficient recruitment over wider gene expression ranges, and improved spatial control over signaling outputs. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.

Fimbrin forms bundles of parallel actin filaments in filopodia, but it remains unclear how fimbrin forms well-ordered bundles. To address this issue, we focused on the cooperative interaction between the actin-binding domain of fimbrin and actin filaments. First, we loosely immobilized actin filaments on a glass surface via a positively charged lipid layer and observed the binding of GFP-fused actin-binding domain 2 of fimbrin using fluorescence microscopy. The actin-binding domain formed low-density clusters with unidirectional growth along actin filaments. When the actin filaments were tightly immobilized to the surface by increasing the charge density of the lipid layer, cluster formation was suppressed. This result suggests that the propagation of cooperative structural changes of actin filaments evoked by binding of the actin-binding domain was suppressed by a strong physical interaction with the glass surface. Interestingly, binding of the fimbrin actin-binding domain shortened the length of loosely immobilized actin filaments. Based on these results, we propose that fimbrin-actin interactions accompanied by unidirectional long-range allostery help the formation of well-ordered parallel actin filament bundles., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The identification of Tunneling Nanotubes (TNTs) and TNT-like structures signified a critical turning point in the field of cell-cell communication. With hypothesized roles in development and disease progression, TNTs' ability to transport biological cargo between distant cells has elevated these structures to a unique and privileged position among other mechanisms of intercellular communication. However, the field faces numerous challenges-some of the most pressing issues being the demonstration of TNTs in vivo and understanding how they form and function. Another stumbling block is represented by the vast disparity in structures classified as TNTs. In order to address this ambiguity, we propose a clear nomenclature and provide a comprehensive overview of the existing knowledge concerning TNTs. We also discuss their structure, formation-related pathways, biological function, as well as their proposed role in disease. Furthermore, we pinpoint gaps and dichotomies found across the field and highlight unexplored research avenues. Lastly, we review the methods employed to date and suggest the application of new technologies to better understand these elusive biological structures., (© 2021 The Authors.)

Actin-rich protrusions are membrane extensions generated by actin polymerization that drive mesenchymal-like cell migration. Characterization of protrusions proteome is crucial for understanding their function. We present a complete step-by-step protocol based on microporous filter-based fractionation of protrusive cellular domains coupled with sample preparation for quantitative proteomics, mass spectrometric data acquisition, and data analysis. This protocol enables purification, quantification, and analysis of the distribution of proteins present in protrusions and cell bodies. For complete details on the use and execution of this protocol, please refer to Dermit et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd 3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

Actin re-organization and degradation of extracellular matrix by metalloproteases (MMPs) facilitate formation of cellular protrusions that are required for cell proliferation and migration. We find that Zn 2+ activation of the Gq-coupled receptor ZnR/GPR39 controls these processes by regulating K + /Cl - co-transporter KCC3, which modulates cell volume. Silencing of KCC3 expression or activity reverses ZnR/GPR39 enhancement of cell proliferation, migration and invasion through Matrigel. Activation of ZnR/GPR39 recruits KCC3 into F-actin rich membrane protrusions, suggesting that it can locally control volume changes. Immunofluorescence analysis indicates that Zn 2+ activation of ZnR/GPR39 and KCC3 are required to enhance formation of F-actin stress fibers and cellular protrusions. In addition, ZnR/GPR39 upregulation of KCC3-dependent transport increases the activity of matrix metalloproteases MMP2 and MMP9. Our study establishes a mechanism in which ZnR/GPR39 orchestrates localization and activation of KCC3, formation of F-actin rich cell protrusions and activation of MMPs, and thereby controls cell proliferation and migration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking., Methods: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo , podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1 . To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics., Results: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro . Srgap1 fl/fl *Six2Cre but not Srgap1 fl/fl *hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1 -knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro . Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS., Conclusions: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases., (Copyright © 2021 by the American Society of Nephrology.)

Actin remodeling is at the heart of the response of cells to external or internal stimuli, allowing a variety of membrane protrusions to form. Fifteen years ago, tunneling nanotubes (TNTs) were identified, bringing a novel addition to the family of actin-supported cellular protrusions. Their unique property as conduits for cargo transfer between distant cells emphasizes the unique nature of TNTs among other protrusions. While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood. In this review, we discuss the role of several actin regulators in the formation of TNTs and suggest potential players based on their comparison with other actin-based protrusions. New perspectives for discovering a distinct TNT formation pathway would enable us to target them in treating the increasing number of TNT-involved pathologies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking., (© 2021 The Author(s).)

The concept of "symmetry breaking" has become a mainstay of modern biology, yet you will not find a definition of this concept specific to biological systems in Wikipedia [...].

Membrane vesicles (MVs) play biologically important roles in Gram-positive bacteria, and purification is essential for their study. Although high-performance flow cytometry has the capability to quantify and isolate specific small particles, it has not been examined for MV isolation. In this study, we used high-performance flow cytometry to analyze MV from Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, prepared by iodixanol density-gradient ultracentrifugation. Analysis of the quality of MV samples before and after sorting showed that the flow cytometric sorting provided higher purity and uniformity compared to gradient isolation alone. The MV purification method using flow cytometry should prove useful for applications requiring a very high purity of MV samples such as proteomic, metagenomic or lipidomic studies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Malignant gliomas including Glioblastoma (GBM) are characterized by extensive diffuse tumor cell infiltration throughout the brain, which represents a major challenge in clinical disease management. While surgical resection is beneficial for patient outcome, it is well recognized that tumor cells at the invasive front or beyond stay behind and constitute a major source of tumor recurrence. Invasive glioma cells also represent a difficult therapeutic target since they are localized within normal functional brain areas with an intact blood brain barrier (BBB), thereby excluding most systemic drug treatments. Cell movement is mediated via the actin cytoskeleton where corresponding membrane protrusions play essential roles. This review provides an overview of the various paths of glioma cell invasion and underlines the specific aspects of the brain microenvironment. We highlight recent insight into tumor microtubes, neuro-glioma synapses and tumor metabolism which can regulate collective invasion processes. We also focus on the deregulation of actin cytoskeleton-related components in the context of glioma invasion, a deregulation that may be controlled by genomic alterations in tumor cells as well as by various external factors, including extracellular matrix (ECM) components and non-malignant stromal cells. Finally we critically assess the challenges and opportunities for therapeutically targeting glioma cell invasion., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5-5) × 10 1 QDs/µm 2 , whereas labeled HER2 expression was higher ranging from (2-10) × 10 2 QDs/µm 2 . Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.

Coordinated directional migration of cells in the mesoderm layer of the early embryo is essential for organization of the body plan. Here we show that mesoderm organization in mouse embryos depends on β-Pix (Arhgef7), a guanine nucleotide exchange factor for Rac1 and Cdc42. As early as E7.5, β-Pix mutants have an abnormally thick mesoderm layer; later, paraxial mesoderm fails to organize into somites. To define the mechanism of action of β-Pix in vivo, we optimize single-cell live-embryo imaging, cell tracking, and volumetric analysis of individual and groups of mesoderm cells. Use of these methods shows that wild-type cells move in the same direction as their neighbors, whereas adjacent β-Pix mutant cells move in random directions. Wild-type mesoderm cells have long polarized filopodia-like protrusions, which are absent in β-Pix mutants. The data indicate that β-Pix-dependent cellular protrusions drive and coordinate collective migration of the mesoderm in vivo.

Cytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Inversely, Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke's previously reported regulation of myosin contractility during dorsal closure., Competing Interests: The authors have declared that no competing interests exist.

Translation of ribosomal protein-coding mRNAs (RP-mRNAs) constitutes a key step in ribosome biogenesis, but the mechanisms that modulate RP-mRNA translation in coordination with other cellular processes are poorly defined. Here, we show that subcellular localization of RP-mRNAs acts as a key regulator of their translation during cell migration. As cells migrate into their surroundings, RP-mRNAs localize to the actin-rich cell protrusions. This localization is mediated by La-related protein 6 (LARP6), an RNA-binding protein that is enriched in protrusions. Protrusions act as hotspots of translation for RP-mRNAs, enhancing RP synthesis, ribosome biogenesis, and the overall protein synthesis in migratory cells. In human breast carcinomas, epithelial-to-mesenchymal transition (EMT) upregulates LARP6 expression to enhance protein synthesis and support invasive growth. Our findings reveal LARP6-mediated mRNA localization as a key regulator of ribosome biogenesis during cell migration and demonstrate a role for this process in cancer progression downstream of EMT., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Collective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, "leader cells" determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Podoplanin and CD44 are transmembrane glycoproteins involved in inflammation and cancer. In this paper, we report that podoplanin is coordinately expressed with the CD44 standard (CD44s) and variant (CD44v) isoforms in vivo-in hyperplastic skin after a pro-inflammatory stimulus with 12- O -tetradecanoylphorbol-13-acetate (TPA)-and in vitro-in cell lines representative of different stages of mouse-skin chemical carcinogenesis, as well as in human squamous carcinoma cell (SCC) lines. Moreover, we identify CD44v10 in the mouse-skin carcinogenesis model as the only CD44 variant isoform expressed in highly aggressive spindle carcinoma cell lines together with CD44s and podoplanin. We also characterized CD44v3-10, CD44v6-10 and CD44v8-10 as the major variant isoforms co-expressed with CD44s and podoplanin in human SCC cell lines. Immunofluorescence confocal microscopy experiments show that these CD44v isoforms colocalize with podoplanin at plasma membrane protrusions and cell-cell contacts of SCC cells, as previously reported for CD44s. Furthermore, CD44v isoforms colocalize with podoplanin in chemically induced mouse-skin SCCs in vivo. Co-immunoprecipitation experiments indicate that podoplanin physically binds to CD44v3-10, CD44v6-10 and CD44v8-10 isoforms, as well as to CD44s. Podoplanin-CD44 interaction is mediated by the transmembrane and cytosolic regions and is negatively modulated by glycosylation of the extracellular domain. These results point to a functional interplay of podoplanin with both CD44v and CD44s isoforms in SCCs and give insight into the regulation of the podoplanin-CD44 association.