Yannick Schwab

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans.

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals.

STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER

Summary Inter-organelle membrane contacts sites (MCSs) are specific subcellular regions favoring the exchange of metabolites and information. We investigated the potential role of the late-endosomal membrane-anchored proteins StAR related lipid transfer domain-3 (STARD3) and STARD3 N-terminal like (STARD3NL) in the formation of MCSs involving late-endosomes (LEs). We demonstrate that both STARD3 and STARD3NL create MCSs between LEs and the endoplasmic reticulum (ER). STARD3 and STARD3NL use a conserved two phenylalanines in an acidic tract (FFAT)-motif to interact with ER-anchored VAP proteins. Together, they form an LE–ER tethering complex allowing heterologous membrane apposition. This LE–ER tethering complex affects organelle dynamics by altering the formation of endosomal tubules. An in situ proximity ligation assay between STARD3, STARD3NL and VAP proteins identified endogenous LE–ER MCS. Thus, we report here the identification of proteins involved in inter-organellar interaction.

Defects in amphiphysin 2 (BIN1) and triads in several forms of centronuclear myopathies

Myotubular myopathy and centronuclear myopathies (CNM) are congenital myopathies characterized by generalized muscle weakness and mislocalization of muscle fiber nuclei. Genetically distinct forms exist, and mutations in BIN1 were recently identified in autosomal recessive cases (ARCNM). Amphiphysins have been implicated in membrane remodeling in brain and skeletal muscle. Our objective was to decipher the pathogenetic mechanisms underlying different forms of CNM, with a focus on ARCNM cases. In this study, we compare the histopathological features from patients with X-linked, autosomal recessive, and dominant forms, respectively, mutated in myotubularin (MTM1), amphiphysin 2 (BIN1), and dynamin 2 (DNM2). We further characterize the ultrastructural defects in ARCNM muscles. We demonstrate that the two BIN1 isoforms expressed in skeletal muscle possess the phosphoinositide-binding domain and are specifically targeted to the triads close to the DHPR–RYR1 complex. Cardiac isoforms do not contain this domain, suggesting that splicing of BIN1 regulates its specific function in skeletal muscle. Immunofluorescence analyses of muscles from patients with BIN1 mutations reveal aberrations of BIN1 localization and triad organization. These defects are also observed in X-linked and autosomal dominant forms of CNM and in Mtm1 knockout mice. In addition to previously reported implications of BIN1 in cancer as a tumor suppressor, these findings sustain an important role for BIN1 skeletal muscle isoforms in membrane remodeling and organization of the excitation–contraction machinery. We propose that aberrant BIN1 localization and defects in triad structure are part of a common pathogenetic mechanism shared between the three forms of centronuclear myopathies.

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development

VIDEO ABSTRACT The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

The glutamate transporter EAAT5 works as a presynaptic receptor in mouse rod bipolar cells.

Membrane neurotransmitter transporters control the concentration of their substrate in the synaptic clefts, through the thermodynamic coupling of uptake to the movement of Na+ and other ions. In addition, excitatory amino acid transporters (EAAT) have a Cl− conductance which is gated by the joint binding of Na+ and glutamate, but thermodynamically uncoupled to the flux of glutamate. This conductance is particularly large in the retina‐specific EAAT5 isoform. In the mouse retina, we located EAAT5 in both cone and rod photoreceptor terminals and in axon terminals of rod bipolar cells. In these later cells, application of glutamate on the axon terminal evoked a current that reversed at ECl, was insensitive to bicuculline, TPMPA, strychnine, dl‐AP5, CNQX and MCPG, but blocked by the glutamate transporter inhibitor dl‐tBOA. Furthermore, short depolarizations of the bipolar cells evoked a dl‐tBOA and Cd2+‐sensitive current whose amplitude was comparable to the glutamate‐evoked current. Its kinetics indicated that EAAT5 was located close to the glutamate release site. For 2 ms depolarizations evoking maximal responses, the EAAT5‐mediated current carried between 2 and 8 times more charge as an average inhibitory GABA or glycine postsynaptic current received spontaneously from amacrine cells, with 10 mm or 0.5 mm intracellular EGTA, respectively. In conditions for which reciprocal inhibition could be monitored, the charge carried by the EAAT5 current was 1.5 times larger than the one carried by the inhibitory postsynaptic currents received from amacrine cells. These results indicate that EAAT5 acts as a major inhibitory presynaptic receptor at mammalian rod bipolar cell axon terminals. This feedback mechanism could control glutamate release at the ribbon synapses of a non‐spiking neuron and increase the temporal contrast in the rod photoreceptor pathway.

A Caenorhabditis elegans model for epithelial–neuronal transdifferentiation

Understanding transdifferentiation—the conversion of one differentiated cell type into another—is important from both basic science and clinical perspectives. In Caenorhabditis elegans, an epithelial cell named Y is initially part of the rectum but later appears to withdraw, migrate, and then become a motor neuron named PDA. Here, we show that this represents a bona fide transdifferentiation event: Y has epithelial hallmarks without detectable neural characteristics, and PDA has no residual epithelial characteristics. Using available mutants and laser microsurgery, we found that transdifferentiation does not depend on fusion with a neighboring cell or require migration of Y away from the rectum, that other rectal epithelial cells are not competent to transdifferentiate, and that transdifferentiation requires the EGL-5 and SEM-4 transcription factors and LIN-12/Notch signaling. Our results establish Y-to-PDA transdifferentiation as a genetically tractable model for deciphering the mechanisms underlying cellular plasticity in vivo.

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.

Luminal signalling links cell communication to tissue architecture during organogenesis

Morphogenesis is the process whereby cell collectives are shaped into differentiated tissues and organs. The self-organizing nature of morphogenesis has been recently demonstrated by studies showing that stem cells in three-dimensional culture can generate complex organoids, such as mini-guts, optic-cups and even mini-brains. To achieve this, cell collectives must regulate the activity of secreted signalling molecules that control cell differentiation, presumably through the self-assembly of microenvironments or niches. However, mechanisms that allow changes in tissue architecture to feedback directly on the activity of extracellular signals have not been described. Here we investigate how the process of tissue assembly controls signalling activity during organogenesis in vivo, using the migrating zebrafish lateral line primordium. We show that fibroblast growth factor (FGF) activity within the tissue controls the frequency at which it deposits rosette-like mechanosensory organs. Live imaging reveals that FGF becomes specifically concentrated in microluminal structures that assemble at the centre of these organs and spatially constrain its signalling activity. Genetic inhibition of microlumen assembly and laser micropuncture experiments demonstrate that microlumina increase signalling responses in participating cells, thus allowing FGF to coordinate the migratory behaviour of cell groups at the tissue rear. As the formation of a central lumen is a self-organizing property of many cell types, such as epithelia and embryonic stem cells, luminal signalling provides a potentially general mechanism to locally restrict, coordinate and enhance cell communication within tissues.

Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses.

Summary With no antiviral drugs or widely available vaccines, Dengue virus (DENV) constitutes a public health concern. DENV replicates at ER-derived cytoplasmic structures that include substructures called convoluted membranes (CMs); however, the purpose of these membrane alterations remains unclear. We determine that DENV nonstructural protein (NS)4B, a promising drug target with unknown function, associates with mitochondrial proteins and alters mitochondria morphology to promote infection. During infection, NS4B induces elongation of mitochondria, which physically contact CMs. This restructuring compromises the integrity of mitochondria-associated membranes, sites of ER-mitochondria interface critical for innate immune signaling. The spatio-temporal parameters of CM biogenesis and mitochondria elongation are linked to loss of activation of the fission factor Dynamin-Related Protein-1. Mitochondria elongation promotes DENV replication and alleviates RIG-I-dependent activation of interferon responses. As Zika virus infection induces similar mitochondria elongation, this perturbation may protect DENV and related viruses from innate immunity and create a favorable replicative environment.