Morphological and Functional Characterization of the Ciliary Pocket by Electron and Fluorescence Microscopy
In many vertebrate cell types, the proximal part of the primary cilium is positioned within an invagination of the plasma membrane known as the ciliary pocket. Recent evidence points to the conclusion that the ciliary pocket comprises a unique site for exocytosis and endocytosis of ciliary proteins, which regulates the spatiotemporal trafficking of receptors into and out of the cilium to control its sensory function. In this chapter, we provide methods based on electron microscopy, 3D reconstruction of fluorescence images as well as live cell imaging suitable for investigating processes associated with endocytosis at the ciliary pocket.
Key wordsPrimary cilia Ciliary pocket Clathrin-dependent endocytosis Early endosomes Cellular signaling
We would like to thank Pierre Bourdoncle (Cellular imaging Facility, Cochin Institute) for his precious help on deconvolution and 3D reconstruction of fluorescence images on RPE1 cells. This work was supported by the University of Copenhagen Excellence Programme for Interdisciplinary Research and the Danish Council for Independent Research (1331-00254).
- 15.Clement CA, Ajbro KD, Koefoed K, Vestergaard ML, Veland IR, Henriques de Jesus MP, Pedersen LB, Benmerah A, Andersen CY, Larsen LA, Christensen ST (2013) TGF-beta signaling is associated with endocytosis at the pocket region of the primary cilium. Cell Rep 3(6):1806–1814. doi: 10.1016/j.celrep.2013.05.020 CrossRefGoogle Scholar
- 16.Molla-Herman A, Ghossoub R, Blisnick T, Meunier A, Serres C, Silbermann F, Emmerson C, Romeo K, Bourdoncle P, Schmitt A, Saunier S, Spassky N, Bastin P, Benmerah A (2010) The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia. J Cell Sci 123(Pt 10):1785–1795. doi: 10.1242/jcs.059519 CrossRefGoogle Scholar
- 18.Tang WB, Ling GH, Sun L, Liu FY (2010) Smad anchor for receptor activation (SARA) in TGF-beta signaling. Front Biosci (Elite Ed) 2:857–860Google Scholar