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Electron Microscopy Core FacilityPublications

Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development.
Goetz, J.G., Steed, E., Ferreira, R.R., Roth, S., Ramspacher, C., Boselli, F., Charvin, G., Liebling, M., Wyart, C., Schwab, Y. & Vermot, J.
Cell Rep. 2014 Mar 13;6(5):799-808. doi: 10.1016/j.celrep.2014.01.032. Epub 2014Feb 20.
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. VIDEO ABSTRACT:
Europe PMC

Correlative Light and Electron Microscopy: From Live Cell Dynamic to 3D Ultrastructure.
Spiegelhalter, C., Laporte, J.F. & Schwab, Y.
Methods Mol Biol. 2014;1117:485-501. doi: 10.1007/978-1-62703-776-1_21.
Correlative light and electron microscopy (CLEM) aims at combining data acquired from the same sample through both imaging modalities. Many combinations can be found in the literature where almost any kind of light microscopy (LM) has been associated to different processing in electron microscopy (EM) and applied to a wide variety of specimen, from cultured cells to multicellular organisms. In this chapter, we focus on a technique that intends to combine LM acquisition on living cells with transmission EM (TEM) analysis. A specific attention is given to the description of a method to bring precise coordinates to the object of interest, to allow a straightforward correlation between LM and EM. Moreover, we describe how, by using high-pressure freezing as a fixation technique, dynamic events observed at the LM are captured and studied at the ultrastructural level.
Europe PMC

Using correlative light and electron microscopy to study zebrafish morphogenesis.
Goetz, J.G., Monduc, F., Schwab, Y. & Vermot, J.
Methods in Molecular Biology (In press).

Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo.
Amoasii, L., Hnia, K., Chicanne, G., Brech, A., Cowling, B.S., Muller, M.M., Schwab, Y., Koebel, P., Ferry, A., Payrastre, B. & Laporte, J.
J Cell Sci. 2013 Apr 15;126(Pt 8):1806-19. doi: 10.1242/jcs.118505. Epub 2013 Feb26.
The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.
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Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.
Lenormand, C., Spiegelhalter, C., Cinquin, B., Bardin, S., Bausinger, H., Angenieux, C., Eckly, A., Proamer, F., Wall, D., Lich, B., Tourne, S., Hanau, D., Schwab, Y., Salamero, J. & de la Salle, H.
PLoS One. 2013;8(4):e60813. doi: 10.1371/journal.pone.0060813. Epub 2013 Apr 5.
Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.
Europe PMC

A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation.
Kolotuev, I., Hyenne, V., Schwab, Y., Rodriguez, D. & Labouesse, M.
Nat Cell Biol. 2013 Feb;15(2):157-68. doi: 10.1038/ncb2662. Epub 2013 Jan 20.
The mechanisms regulating the extension of small unicellular tubes remain poorly defined. Here we identify several steps in Caenorhabditis elegans excretory canal growth, and propose a model for lumen extension. Our results suggest that the basal and apical excretory membranes grow sequentially: the former extends first like an axon growth cone; the latter extends next as a result of an osmoregulatory activity triggering peri-apical vesicles (a membrane reservoir) to fuse with the lumen. An apical cytoskeletal web including intermediate filaments and actin crosslinking proteins ensures straight regular lumen growth. Expression of several genes encoding proteins mediating excretory lumen extension, such as the osmoregulatory STE20-like kinase GCK-3 and the intermediate filament IFB-1, is regulated by ceh-26 (here referred to as pros-1), which we found essential for excretory canal formation. Interestingly, PROS-1 is homologous to vertebrate Prox1, a transcription factor controlling lymphatic vessel growth. Our findings have potential evolutionary implications for the origin of fluid-collecting organs, and provide a reference for lymphangiogenesis.
Europe PMC

Selective autophagy degrades DICER and AGO2 and regulates miRNA activity.
Gibbings, D., Mostowy, S., Jay, F., Schwab, Y., Cossart, P. & Voinnet, O.
Nat Cell Biol. 2012 Dec;14(12):1314-21.
MicroRNAs (miRNAs) form a class of short RNAs ( approximately 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.
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The BAR Domain Protein Arfaptin-1 Controls Secretory Granule Biogenesis at the trans-Golgi Network.
Gehart, H., Goginashvili, A., Beck, R., Morvan, J., Erbs, E., Formentini, I., De Matteis, M.A., Schwab, Y., Wieland, F.T. & Ricci, R.
Dev Cell. 2012 Oct 16;23(4):756-68. doi: 10.1016/j.devcel.2012.07.019. Epub 2012Sep 13.
BAR domains can prevent membrane fission through their ability to shield necks of budding vesicles from fission-inducing factors. However, the physiological role of this inhibitory function and its regulation is unknown. Here we identify a checkpoint involving the BAR-domain-containing protein Arfaptin-1 that controls biogenesis of secretory granules at the trans-Golgi network (TGN). We demonstrate that protein kinase D (PKD) phosphorylates Arfaptin-1 at serine 132, which disrupts the ability of Arfaptin-1 to inhibit the activity of ADP ribosylation factor, an important component of the vesicle scission machinery. The physiological significance of this regulatory mechanism is evidenced by loss of glucose-stimulated insulin secretion due to granule scission defects in pancreatic beta cells expressing nonphosphorylatable Arfaptin-1. Accordingly, depletion of Arfaptin-1 leads to the generation of small nonfunctional secretory granules. Hence, PKD-mediated Arfaptin-1 phosphorylation is necessary to ensure biogenesis of functional transport carriers at the TGN in regulated secretion.
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In vivo visualization of delta opioid receptors upon physiological activation uncovers a distinct internalization profile.
Faget, L., Erbs, E., Le Merrer, J., Scherrer, G., Matifas, A., Benturquia, N., Noble, F., Decossas, M., Koch, M., Kessler, P., Vonesch, J.L., Schwab, Y., Kieffer, B.L. & Massotte, D.
J Neurosci. 2012 May 23;32(21):7301-10.
G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.
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Mouse delta opioid receptors are located on presynaptic afferents to hippocampal pyramidal cells.
Rezai, X., Faget, L., Bednarek, E., Schwab, Y., Kieffer, B.L. & Massotte, D.
Cell Mol Neurobiol. 2012 May;32(4):509-16. Epub 2012 Jan 18.
Delta opioid receptors participate in the control of chronic pain and emotional responses. Recent data have also identified their implication in drug-context associations pointing to a modulatory role on hippocampal activity. We used fluorescent knock-in mice that express a functional delta opioid receptor fused at its carboxy terminus with the green fluorescent protein in place of the native receptor to investigate the receptor neuroanatomical distribution in this structure. Fine mapping of the pyramidal layer was performed in hippocampal acute brain slices and organotypic cultures using fluorescence confocal imaging, co-localization with pre- and postsynaptic markers and correlative light-electron microscopy. The different approaches concurred to identify delta opioid receptors on presynaptic afferents to glutamatergic principal cells. In the latter, only scarce receptors were detected that were confined within the Golgi or vesicular intracellular compartments with no receptor present at the cell surface. In the mouse hippocampus, expression of functional delta opioid receptors is therefore mostly associated with interneurons emphasizing a presynaptic modulatory effect on the pyramidal cell firing rate.
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Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes.
Gibbings, D., Leblanc, P., Jay, F., Pontier, D., Michel, F., Schwab, Y., Alais, S., Lagrange, T. & Voinnet, O.
Nat Struct Mol Biol. 2012 Apr 8;19(5):517-24, S1. doi: 10.1038/nsmb.2273.
Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrP(C) contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrP(C) preferentially binds AGO, and PrP(C) promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrP(C). We propose that dynamic interactions between PrP(C)-enriched endosomes and subcellular foci of AGO underpin these effects.
Europe PMC

Expression of exocytosis proteins in rat supraoptic nucleus neurones.
Tobin, V., Schwab, Y., Lelos, N., Onaka, T., Pittman, Q.J. & Ludwig, M.
J Neuroendocrinol. 2012 Apr;24(4):629-41. doi: 10.1111/j.1365-2826.2011.02237.x.
In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca(2+) -dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory.
Europe PMC

Targeted ultramicrotomy: a valuable tool for correlated light and electron microscopy of small model organisms.
Kolotuev, I., Bumbarger, D.J., Labouesse, M. & Schwab, Y.
Methods Cell Biol. 2012;111:203-22.
Correlative light and electron microscopy (CLEM) is used when one needs to combine both imaging modalities on the same sample. When working on living small model organisms, such as Caenorhabditis elegans, specific CLEM protocols are required to acquire high-resolution light microscopic images of a region of interest and thereafter to relocate and study the same object at the ultrastructural level using a transmission electron microscope. In this chapter, we describe how to process living specimens from the confocal microscope to the transmission electron microscopy (TEM), focusing on an improved ultramicrotomy technique that allows a precise and reliable targeting of the object of interest. This improvement significantly reduces the time consuming and frequently frustrating search for the region of interest. Our targeted ultramicrotomy protocol is versatile enough to be applied on a variety of bulk specimens, such as fly and fish embryos, or mouse tissues.
Europe PMC

The podocyte protein nephrin is required for cardiac vessel formation.
Wagner, N., Morrison, H., Pagnotta, S., Michiels, J.F., Schwab, Y., Tryggvason, K., Schedl, A. & Wagner, K.D.
Hum Mol Genet. 2011 Jun 1;20(11):2182-94. Epub 2011 Mar 14.
Nephrin (NPHS1) has been described as an important structural protein of kidney podocytes. Mutations in this gene lead to the Finnish-type congenital nephrotic syndrome. More recently, a role of nephrin as a signalling molecule in kidney podocytes has been identified. Here, we show that nephrin not only has a function in kidney podocytes, but is also required for cardiovascular development. Nephrin is expressed in the epicardium and coronary vessels during human and mouse embryonic development. Nephrin knockout embryos showed abnormal epicardial cell morphology and, at later stages of development, a reduced number of coronary vessels due to increased apoptosis, and in addition, cardiac fibrosis. Connexin 43, which is required for coronary vessel formation, was downregulated in nephrin knockout embryos. Expression of the p75NTR neurotrophin receptor, a known mediator of apoptosis, was increased in mutants. Furthermore, co-immunoprecipitation studies demonstrated a direct interaction of nephrin with p75NTR. Primary nephrin-deficient cardiac cells showed a 5-fold higher rate of apoptosis in response to progenitor of nerve growth factor compared with wild-type cells, which could be rescued by RNAi against p75NTR. Taken together, our data demonstrate that nephrin directly interacts with p75NTR and reveal an important role for nephrin in murine cardiac development by permitting survival of cardiovascular progenitor cells.
Europe PMC

PAT-12, a potential anti-nematode target, is a new spectraplakin partner essential for Caenorhabditis elegans hemidesmosome integrity and embryonic morphogenesis.
Hetherington, S., Gally, C., Fritz, J.A., Polanowska, J., Reboul, J., Schwab, Y., Zahreddine, H., Behm, C. & Labouesse, M.
Dev Biol. 2011 Feb 15;350(2):267-78. Epub 2010 Dec 3.
Caenorhabditis elegans embryonic elongation depends on both epidermal and muscle cells. The hemidesmosome-like junctions, commonly called fibrous organelles (FOs), that attach the epidermis to the extracellular matrix ensure muscle anchoring to the cuticular exoskeleton and play an essential role during elongation. To further define how hemidesmosomes might control elongation, we searched for factors interacting with the core hemidesmosome component, the spectraplakin homolog VAB-10. Using the VAB-10 plakin domain as bait in a yeast two-hybrid screen, we identified the novel protein T17H7.4. We also identified T17H7.4 in an independent bioinformatic search for essential nematode-specific proteins that could define novel anti-nematode drug or vaccine targets. Interestingly, T17H7.4 corresponds to the C. elegans equivalent of the parasitic OvB20 antigen, and has a characteristic hemidesmosome distribution. We identified two mutations in T17H7.4, one of which defines the uncharacterized gene pat-12, previously identified in screens for genes required for muscle assembly. Using isoform-specific GFP constructs, we showed that one pat-12 isoform with a hemidesmosome distribution can rescue a pat-12 null allele. We further found that lack of pat-12 affects hemidesmosome integrity, with marked defects at the apical membrane. PAT-12 defines a novel component of C. elegans hemidesmosomes, which is required for maintaining their integrity. We suggest that PAT-12 helps maintaining VAB-10 attachment with matrix receptors.
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Defects in amphiphysin 2 (BIN1) and triads in several forms of centronuclear myopathies.
Toussaint, A., Cowling, B.S., Hnia, K., Mohr, M., Oldfors, A., Schwab, Y., Yis, U., Maisonobe, T., Stojkovic, T., Wallgren-Pettersson, C., Laugel, V., Echaniz-Laguna, A., Mandel, J.L., Nishino, I. & Laporte, J.
Acta Neuropathol. 2011 Feb;121(2):253-66. Epub 2010 Oct 7.
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.
Europe PMC

Mammalian retinal horizontal cells are unconventional GABAergic neurons.
Deniz, S., Wersinger, E., Schwab, Y., Mura, C., Erdelyi, F., Szabo, G., Rendon, A., Sahel, J.A., Picaud, S. & Roux, M.J.
J Neurochem. 2011 Feb;116(3):350-62. doi: 10.1111/j.1471-4159.2010.07114.x. Epub2010 Dec 13.
Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.
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Reevaluation of dystrophin localization in the mouse retina.
Wersinger, E., Bordais, A., Schwab, Y., Sene, A., Benard, R., Alunni, V., Sahel, J.A., Rendon, A. & Roux, M.J.
Invest Ophthalmol Vis Sci. 2011 Oct 7;52(11):7901-8. doi: 10.1167/iovs.11-7519.Print 2011.
PURPOSE. The roles of dystrophins in retinal physiology remain elusive. The lack of proper clustering of the potassium channel Kir4.1 and of the aquaporin AQP4 was proposed to be the basis of the ERG abnormality observed in many Duchenne muscular dystrophy (DMD) patients. However, the electroretinogram of Dp71-null mice, in which this clustering is disrupted, shows only a moderate reduction of the b-wave with no change in the implicit times. Additionally, the deficit in color discrimination found in DMD patients is hard to explain through the known expression of DMD gene products. The authors thus decided to reexamine their distribution in the mouse retina. METHODS. Messenger RNA distribution was assessed by PCR coupled to laser microdissection of the outer and inner nuclear layers and by in situ hybridization for Dp427. Mouse retinas were double labeled for dystrophins versus presynaptic and postsynaptic proteins or antibodies specific for Dp427 or Dp427+Dp260. RESULTS. Messengers for Dp427, Dp260, and Dp140 were present in the inner nuclear layer. Dp427 mRNA was further detected in bipolar cells and in some amacrine cells by in situ hybridization. Comparative labeling in wild-type and mdx(5Cv) retinas (lacking Dp427) indicated a differential distribution of Dp427 and Dp260 between rod and cone terminals. CONCLUSIONS. In addition to their localization in photoreceptor terminals, Dp427, Dp260, and Dp140 are expressed in inner nuclear layer neurons, notably in bipolar cells for Dp427. Dp427 was proportionally more expressed in cone- than in rod-associated synapses compared with Dp260.
Europe PMC

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms.
Kolotuev, I., Schwab, Y. & Labouesse, M.
Biol Cell. 2009 Dec 4;102(2):121-32.
BACKGROUND INFORMATION: CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). RESULTS: We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron-microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high-pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 mum distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non-correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron-microscopic protocols, making them more accessible to a wider range of researchers. CONCLUSIONS: We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time-demanding procedure of specimen preparation for any kind of TEM.
Europe PMC

From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.
Spiegelhalter, C., Tosch, V., Hentsch, D., Koch, M., Kessler, P., Schwab, Y. & Laporte, J.
PLoS One. 2010 Feb 3;5(2):e9014.
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.
Europe PMC

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.
Buj-Bello, A., Fougerousse, F., Schwab, Y., Messaddeq, N., Spehner, D., Pierson, C.R., Durand, M., Kretz, C., Danos, O., Douar, A.M., Beggs, A.H., Schultz, P., Montus, M., Denefle, P. & Mandel, J.L.
Hum Mol Genet. 2008 Jul 15;17(14):2132-43. Epub 2008 Apr 22.
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.
Europe PMC

A Caenorhabditis elegans model for epithelial-neuronal transdifferentiation.
Jarriault, S., Schwab, Y. & Greenwald, I.
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3790-5. Epub 2008 Feb 28.
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.
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DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1.
Lecroisey, C., Martin, E., Mariol, M.C., Granger, L., Schwab, Y., Labouesse, M., Segalat, L. & Gieseler, K.
Mol Biol Cell. 2008 Mar;19(3):785-96. Epub 2007 Dec 19.
In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.
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Endogenous modulators of synaptic transmission: cannabinoid regulation in the supraoptic nucleus.
McDonald, N.A., Kuzmiski, J.B., Naderi, N., Schwab, Y. & Pittman, Q.J.
Prog Brain Res. 2008;170:129-36.
The magnocellular neurons of the hypothalamic supraoptic nucleus (SON) are a major source of both systemic and central release of the neurohypophyseal peptides, oxytocin (OXT) and arginine-vasopressin (AVP). Both OXT and AVP are released from the somatodendritic compartment of magnocellular neurons and act within the SON to modulate the electrophysiological function of these cells. Cannabinoids (CBs) affect hormonal output and the SON may represent a neural substrate through which CBs exert specific physiological and behavioural effects. Dynamic modulation of synaptic inputs is a fundamental mechanism through which neuronal output is controlled. Dendritically released OXT acts on autoreceptors to generate endocannabinoids (eCBs) which modify both excitatory and inhibitory inputs to OXT neurons through actions on presynaptic CB receptors. As such, OXT and eCBs cooperate to shape the electrophysiological properties of magnocellular OXT neurons, regulating the physiological function of this nucleus. Further study of eCB signalling in the SON, including its interaction with AVP neurons, promises to extend our understanding of the synaptic regulation of SON physiological function.
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The glutamate transporter EAAT5 works as a presynaptic receptor in mouse rod bipolar cells.
Wersinger, E., Schwab, Y., Sahel, J.A., Rendon, A., Pow, D.V., Picaud, S. & Roux, M.J.
J Physiol. 2006 Nov 15;577(Pt 1):221-34. Epub 2006 Sep 14.
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 E(Cl), 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 Cd(2+)-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.
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The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans.
Liegeois, S., Benedetto, A., Garnier, J.M., Schwab, Y. & Labouesse, M.
J Cell Biol. 2006 Jun 19;173(6):949-61.
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.
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Physiological maturation of photoreceptors depends on the voltage-gated sodium channel NaV1.6 (Scn8a).
Cote, P.D., De Repentigny, Y., Coupland, S.G., Schwab, Y., Roux, M.J., Levinson, S.R. & Kothary, R.
J Neurosci. 2005 May 18;25(20):5046-50.
Voltage-gated sodium channels (VGSCs) ensure the saltatory propagation of action potentials along axons by acting as signal amplifiers at the nodes of Ranvier. In the retina, activity mediated by VGSCs is important for the refinement of the retinotectal map. Here, we conducted a full-field electroretinogram (ERG) study on mice null for the sodium channel NaV1.6. Interestingly, the light-activated hyperpolarization of photoreceptor cells (the a-wave) and the major "downstream" components of the ERG, the b-wave and the oscillatory potentials, are markedly reduced and delayed in these mice. The functional deficit was not associated with any morphological abnormality. We demonstrate that Scn8a is expressed in the ganglion and inner nuclear layers and at low levels in the outer nuclear layer beginning shortly before the observed ERG deficit. Together, our data reveal a previously unappreciated role for VGSCs in the physiological maturation of photoreceptors.
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Dendritically released transmitters cooperate via autocrine and retrograde actions to inhibit afferent excitation in rat brain.
Hirasawa, M., Schwab, Y., Natah, S., Hillard, C.J., Mackie, K., Sharkey, K.A. & Pittman, Q.J.
J Physiol. 2004 Sep 1;559(Pt 2):611-24. Epub 2004 Jul 14.
Oxytocin is released from supraoptic magnocellular neurones and is thought to act at presynaptic receptors to inhibit transmitter release. We now show that this effect is mediated by endocannabinoids, but that oxytocin nonetheless plays an important role in endocannabinoid signalling. WIN55,212-2, a cannabinoid receptor agonist, mimicked the action of oxytocin and occluded oxytocin-induced presynaptic inhibition. The cannabinoid action is at the presynaptic terminal as shown by alteration in paired pulse ratio, a reduction in miniature EPSC frequency and immunohistochemical localization of CB1 receptors on presynaptic terminals. AM251, a CB1 receptor antagonist, blocked both the WIN55,212-2 and the oxytocin-induced presynaptic inhibition of EPSCs. Depolarization of postsynaptic magnocellular neurones (which contain fatty acid amide hydrolase, a cannabinoid catabolic enzyme) caused a transient inhibition of EPSCs that could be blocked by both the AM251 and Manning compound, an oxytocin/vasopressin receptor antagonist. This indicates that somatodendritic peptide release and action on previously identified autoreceptors facilitates the release of endocannabinoids that act as mediators of presynaptic inhibition.
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Expression of tetrodotoxin-sensitive and resistant sodium channels by rat melanotrophs.
Schwab, Y., Jahke, R. & Jover, E.
Neuroreport. 2004 May 19;15(7):1219-23.
Rat melanotrophs fire Na+ and Ca2(+)-dependent action potentials. Whereas the molecular identity of Ca2+ channels expressed by these cells is well documented, less is known about Na channels. We characterize the expression of seven sodium channel alpha-subunit and the beta1- and beta2-subunit mRNAs. The tetrodotoxin-resistant Nav1.8 and Nav1.9 alpha subunit mRNAs are detected in the newborn intermediate lobe and in cultured melanotrophs. Electrophysiological recordings further demonstrate the expression of both tetrodotoxin-sensitive and tetrodotoxin-resistant currents by dissociated melanotrophs. Moreover, activated sodium channels are able to elicit intracellular calcium waves, both in the absence or in the presence of tetrodotoxin. This work shows that rat melanotrophs express functional tetrodotoxin-resistant sodium channels, whose activation can lead to the generation of intracellular calcium waves.
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Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones.
Schwab, Y., Mouton, J., Chasserot-Golaz, S., Marty, I., Maulet, Y. & Jover, E.
Brain Res Mol Brain Res. 2001 Nov 30;96(1-2):1-13.
In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation. Synaptotagmins, a family of Ca(2+)-sensor proteins that includes neurone-specific members involved in regulating neurotransmitter exocytosis, are among the molecules that can tune the fusion mechanism. Using immunocytochemistry, confocal and electron microscopy, the localisation of synaptotagmin I in the dendrites of cultured rat hypothalamic neurones was demonstrated. Synaptotagmin labelling is concentrated at dendritic branch points, and in microprocesses. Following depolarisation, the N-terminal domain of synaptotagmin was detected at the extracellular surface of the dendritic plasma membrane. The insertion of synaptotagmin in the plasma membrane was elicited by L-type Ca(2+) channel activation and by mobilisation of the internal ryanodine-sensitive Ca(2+)stores. Furthermore, the localisation of L-type Ca(2+) channels and of ryanodine receptors, relative to the localisation of synaptotagmin in dendrites, suggests that both Ca(2+) entry and intracellular Ca(2+) stores may contribute to the fusion of dendritic transport vesicles with the membrane. Fusion is likely to involve SNAP-25 and syntaxin 1 as both proteins were also identified in dendrites. Taken together these results suggest a putative regulatory role of synaptotagmins in the membrane fusion events that contribute to shaping the dendritic tree during development.
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