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Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.

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. The architecture of functional TNTs is still under debate. Here, the authors combine correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structure of TNTs in neuronal cells, showing that they form structures that are distinct form other membrane protrusions.

Key Points Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane. They act as sites of attachment to — and degradation of — the extracellular matrix. These structures contain actin regulators such as cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), adaptor proteins such as Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and several pericellular proteases. Podosomes are found in vascular smooth muscle and endothelial cells, as well as in cells derived from monocyte lineages. Their presence correlates with migratory ability. Invadopodia are found in invasive human cancer cells. In two-dimensional culture, their presence correlates with invasive behaviour. However, in three-dimensional culture and in vivo , invadopodium-associated proteins are also required for cell growth. Podosome-associated proteins have been implicated in human developmental and immune disorders, and dysregulation of podosome formation is associated with atherosclerosis. Small-molecule regulation of podosomes and invadopodia might represent a new therapeutic strategy to treat several diseases. Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. Progress has been made in our understanding of the regulation and function of these structures, and their role in human disease. Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to — and degradation of — the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott–Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

“Frustrated” coated pits associate with collagen fibers and appear to promote cell adhesion in three-dimensional collagen networks. Clathrin-coated pits are well known to be involved in receptor-mediated endocytosis. Independent of their role in endocytosis, Elkhatib et al. observed that clathrin-coated structures strongly accumulated along collagen fibers in migrating cells. Clathrin-coated structures assembled on and then partially wrapped around and pinched the fibers. In a three-dimensional (3D) network, this mechanism provided multiple anchoring points along cellular protrusions. In the absence of clathrin-coated structures, protrusions were shorter and migration was impaired. This mode of adhesion may cooperate with classical focal adhesions to help cancer cells move in a 3D environment. Science , this issue p. eaal4713 Migrating cells often use focal adhesions in order to move. Focal adhesions are less prominent in cells migrating in three-dimensional (3D) as compared with 2D environments. We looked for alternative adhesion structures supporting cell migration. We analyzed the dynamics of clathrin-coated pits in cells migrating in a 3D environment of collagen fibers. Both topological cues and local engagement of integrins triggered the accumulation of clathrin-coated structures on fibers. Clathrin/adaptor protein 2 (AP-2) lattices pinched collagen fibers by adopting a tube-like morphology and regulated adhesion to fibers in an endocytosis-independent manner. During migration, tubular clathrin/AP-2 lattices stabilized cellular protrusions by providing anchoring points to collagen fibers. Thus, tubular clathrin/AP-2 lattices promote cell adhesion that, in coordination with focal adhesions, supports cell migration in 3D.

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

Transmission of HIV-1 via intercellular connections has been estimated as 100–1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies 1 , 2 . Such effective intercellular transfer of HIV-1 could occur through virological synapses 3 , 4 , 5 or target-cell filopodia connected to infected cells 6 . Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells 7 , 8 , 9 , 10 , 11 , 12 , 13 , and allow calcium-mediated signals to spread between connected myeloid cells 9 . However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types 7 , 12 , as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.

Tumor cells can engage in a process called collective invasion, in which cohesive groups of cells invade through interstitial tissue. Here, we identified an epigenetically distinct subpopulation of breast tumor cells that have an enhanced capacity to collectively invade. Analysis of spheroid invasion in an organotypic culture system revealed that these \"trailblazer\" cells are capable of initiating collective invasion and promote non-trailblazer cell invasion, indicating a commensal relationship among subpopulations within heterogenous tumors. Canonical mesenchymal markers were not sufficient to distinguish trailblazer cells from non-trailblazer cells, suggesting that defining the molecular underpinnings of the trailblazer phenotype could reveal collective invasion-specific mechanisms. Functional analysis determined that DOCK10, ITGA11, DAB2, PDFGRA, VASN, PPAP2B, and LPAR1 are highly expressed in trailblazer cells and required to initiate collective invasion, with DOCK10 essential for metastasis. In patients with triple-negative breast cancer, expression of these 7 genes correlated with poor outcome. Together, our results indicate that spontaneous conversion of the epigenetic state in a subpopulation of cells can promote a transition from in situ to invasive growth through induction of a cooperative form of collective invasion and suggest that therapeutic inhibition of trailblazer cell invasion may help prevent metastasis.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

The cell surface of Trichomonas vaginalis , causative agent of human trichomoniasis, plays a pivotal role in parasite adhesion, motility, and intercellular communication. Scanning electron microscopy (SEM) is widely used to explore the surface projections involved in these processes; however, standard sample preparation via critical point drying (CPD) often damages delicate membrane projections. Here, we optimized a hexamethyldisilazane (HMDS) drying protocol as a reliable alternative to CPD for ultrastructural analysis of T. vaginalis using SEM. Both drying methods were compared in terms of image quality and artifact formation. HMDS drying significantly improved the preservation of fragile projections, such as filopodia-, cytoneme-, and lamellipodia-like structures, compared to CPD. Our results show that susceptibility to CPD-induced artifacts may vary among highly adherent T. vaginalis strains, highlighting the need for caution in SEM interpretation. In a strain previously CPD-characterized by exhibiting a low number of cytonemes, quantitative analyses revealed a marked increase in the number of parasites with filopodia and cytonemes upon HMDS, accompanied by a reduction in drying-associated artifacts. In contrast, other strains exhibited similar quantitative results for both methods, though HMDS demonstrated a slight qualitative enhancement. HMDS drying also enabled the identification of novel ultrastructural features of T. vaginalis , including (a) long (>20 µm) cytonemes forming network-like connections between parasites and host cells, and (b) thin posterior and axostylar cytoneme-like structures that seems involved in host cell adhesion. Moreover, HMDS provided better morphological preservation of amoeboid forms and enhanced visualization of parasite–host interactions, revealing membranous networks not previously observed with CPD. Altogether, this study demonstrates the importance of the drying methods for sample preparation and expands the approaches for parasite imaging, revealing HMDS as a valuable option that could provide new ultrastructural insights into the surface morphology and intercellular communication mechanisms of T. vaginalis.