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.
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.
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.