Search

International audience; 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.

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.

Arf6-dependent membrane dynamics concentrates active Cdc42 at the leading edge of migrating cells., Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6–aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.

International audience; Heparan sulfate proteoglycan receptor syndecan-1 interacts with the carboxyl-terminal LG4/5 domain in laminin 332 (alpha3LG4/5) and participates in cell adhesion and spreading. To dissect the function of syndecan-1 in these processes, we made use of a cell adhesion model in which syndecan-1 exclusively interacts with a recombinantly expressed alpha3LG4/5 fragment. Plating HT1080 cells on this fragment induces the formation of actin-containing protrusive structures in an integrin-independent manner. Here we show that syndecan-1-mediated formation of membrane protrusions requires dephosphorylation of tyrosine residues in syndecan-1. Accordingly, inhibition of phosphatases with orthovanadate decreases cell adhesion to the alpha3LG4/5 fragment. We demonstrate that the PDZ-containing protein syntenin-1, known to connect cytoskeletal proteins, binds to syndecan-1 in cells plated on the alpha3LG4/5 fragment and participates in the formation of membrane protrusions. We further show that syntenin-1 recruitment depends on the dephosphorylation of Tyr-309 located within syndecan-1 PDZ binding domain EFYA. We propose that tyrosine dephosphorylation of syndecan-1 may regulate its association with cytoskeleton components.

APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

International audience; The ultrastructure of the mature spermatozoon of Gyliauchen sp., a parasite of the dusky rabbitfish Siganus fuscescens, was studied by transmission electron microscopy. The spermatozoon possesses two axonemes of the 9+"1" pattern of Trepaxonemata, four attachment zones, one mitochondrion, a nucleus, cortical microtubules, external ornamentation of the plasma membrane, and spine-like bodies. The main characteristics of this spermatozoon are the presence of one mitochondrion, spine-like bodies not associated with the external ornamentation, and a posterior extremity of type 3 that is characterized by the following sequence: posterior extremity of the nucleus then posterior extremity of the second axoneme. Numerous other ultrastructural features are also discussed and compared to the digenean spermatology literature. This is the first study of a member of the Gyliauchenidae and the fourth within the Lepocreadioidea. The results show that many ultrastructural characters are variable within this superfamily and could be useful for phylogeny.

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.

International audience; Proteolytic degradation of the extracellular matrix (ECM) is one intrinsic property of metastatic tumor cells to breach tissue barriers and to disseminate into different tissues. This process is initiated by the formation of invadopodia, which are actin-driven, finger-like membrane protrusions. Yet, little is known on how invadopodia are endowed with the functional machinery of proteolytic enzymes [1, 2]. The key protease MT1-MMP (membrane type 1-matrix metalloproteinase) confers proteolytic activity to invadopodia and thus invasion capacity of cancer cells [3-6]. Here, we report that MT1-MMP-dependent matrix degradation at invadopodia is regulated by the v-SNARE TI-VAMP/VAMP7, hence providing the molecular inventory mediating focal degradative activity of cancer cells. As observed by TIRF microscopy, MT1-MMP-mCherry and GFP-VAMP7 were simultaneously detected at proteolytic sites. Functional ablation of VAMP7 decreased the ability of breast cancer cells to degrade and invade in a MT1-MMP-dependent fashion. Moreover, the number of invadopodia was dramatically decreased in VAMP7- and MT1-MMP-depleted cells, indicative of a positive-feedback loop in which the protease as a cargo of VAMP7-targeted transport vesicles regulates maturation of invadopodia. Collectively, these data point to a specific role of VAMP7 in delivering MT1-MMP to sites of degradation, maintaining the functional machinery required for invasion.

International audience; Transforming growth factor beta (TGF-beta) has a strong impact on liver development and physiopathology, exercised through its pleiotropic effects on growth, differentiation, survival, and migration. When exposed to TGF-beta, the mhAT3F cells, immortalized, highly differentiated hepatocytes, maintained their epithelial morphology and underwent dramatic alterations of adhesion, leading to partial or complete detachment from a culture plate, followed by readhesion and spreading. These alterations of adhesive behavior were caused by sequential changes in expression of the alpha5beta1 integrin and of its ligand, the fibronectin. The altered specificity of anchorage to the extracellular matrix gave rise to changes in cells' collective motility: cohorts adhering to fibronectin maintained a persistent, directional motility, with ezrin-rich pathfinder cells protruding from the tips of the cohorts. The absence of adhesion to fibronectin prevented the appearance of polarized pathfinders and lead to random, oscillatory motility. Our data suggest a novel role for TGF-beta in the control of collective migration of epithelial cohorts.