Resolving septin functions in cell polarity and primary cilia
- Septins are a conserved family of polymerizing GTP/GDP-binding proteins that were discovered in budding yeast as the scaffold for cytokinesis machinery and required for polarized membrane growth. In mammalian cells, they play diverse roles in cytokinesis, cell morphogenesis and as scaffolds and diffusion barriers. Septins are also implicated in many human diseases including cancer and neuropathies. In epithelial cells, formation and maintenance of cell polarity depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. We show that in epithelial cells septin 2 (SEPT2) fibers colocalize with polyglutamylated (polyGlu) microtubule tracks where vesicles containing apical or basolateral proteins exit, and that SEPT2 maintains polyGlu-microtubule tracks and impedes the binding of tubulin to microtubule-associated protein 4 (MAP4) to facilitate vesicle transport. SEPT2 depletion gives rise to intracellular accumulation of apical and basolateral membrane proteins, a loss of polyGlu-microtubules, and fibroblast-shaped cells. Therefore, SEPT2 is required for polarized epithelia biogenesis. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. We show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics, and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF wash-out. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin-depletion by siRNA. FCF would be a promising tool to study mammalian septin organization and functions. In animal cells, the primary cilium transduces extracellular signals through signaling receptors localized in the ciliary membrane, but how these ciliary membrane proteins are retained in the cilium is unknown. We found that ciliary membrane proteins were highly mobile, but their diffusion was impeded at the base of the cilium by a diffusion barrier. SEPT2 localized at the base of the ciliary membrane. SEPT2 depletion resulted in loss of ciliary membrane protein localization and Sonic hedgehog signal transduction, and inhibited ciliogenesis. Thus, SEPT2 is part of a diffusion barrier at the base of the ciliary membrane and is essential for retaining receptor-signaling pathways in the primary cilium. Having demonstrated that there is a diffusion barrier at the base of the primary cilium and ciliary membrane protein is highly mobile in the ciliary membrane, I sought to determine the unknown dynamics and mechanism of ciliary membrane proteins trafficking on ciliary membrane at single molecule level. Here, I used monovalent streptavidin-conjugated fluorophores to image a single ciliary membrane receptor on the ciliary membrane of live cell and resolve their dynamics. The ciliary membrane receptor appears to travel on ciliary membrane by intraflagellar trafficking (IFT) and more efforts are being devoted into studying the mechanisms for ciliary membrane trafficking.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Department of Biology.
|Nelson, W. J. (W. James)
|Nelson, W. J. (W. James)
|Statement of responsibility
|Submitted to the Department of Biology.
|Ph. D. Stanford University 2012
- © 2011 by Qicong Hu
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