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:
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.
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.
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.