Year: 2008
Type: dissertation
Abstract: Cilia are microtubule containing, hair-like structures protruding from the membrane of most cell types. Recent discoveries have linked cilia to several signal transduction pathways, including Wnt and Hedgehog signaling. Loss of normal ciliary function in mammals is associated with multiple disorders, including retinitis pigmentosa, neurodegeneration, disturbances of left-to-right asymmetry and cystic kidney disease. Structurally, cilia consist of a microtubule-based axoneme, covered with a specialized plasma membrane. Cilia anchor at the basal body and are separated from the rest of the cell body by a terminal plate at the distal end of the basal body, a so called transition zone, restricting protein entry into the cilium. A large number of intraflagellar transport (IFT) molecules have been characterized, which orchestrate a protein transport within the cilium but little is known about the specific transport mechanisms into the ciliary compartment. In this study we generated polyclonal cell-lines of canine kidney epithelial cells, stably expressing ciliary proteins tagged with a modified variant of the yellow fluorescent protein. This enabled us to observe dynamically the translocation of the proteins into the primary cilium in live cells. As model ciliary proteins, we chose the microtubule-associated protein EB1, and two transmembrane receptors: Kim1 and SmoA1 (a constitutively-active form of Smoothened). To address the question of how proteins get into the cilium and which trafficking routes they follow, we developed a ciliary FRAP protocol. This method is based on the quantification of fluorescence recovery in the cilium after complete photobleaching of the ciliary shaft. Recovery of fluorescence represents the transport of newly arrived ciliary protein. Ciliary FRAP allows to make indirect judgments about the transport mechanisms specific for ciliary proteins. We show a discrepancy in kinetic characteristics for all studied ciliary proteins. Treatment with Brefeldin A, chemical that blocks vesicular assembly at Golgi apparatus, significantly delayed the transport of Smoothened into the ciliary compartment, without affecting the trafficking characteristics of other molecules. These data suggest the existence of diversified transport routes for ciliary proteins that have not been shown before. To further elucidate the molecular mechanism of the transport we studied involvement of Rab proteins in the trafficking of ciliary proteins. To reduce cytotoxicity of Rab GTPases locked in inactive form we implemented a tetracyclineinducible lentiviral system. We showed that Rab8A, a GTPase involved in a membrane trafficking from the Golgi to the basolateral membrane, is localized to the primary cilium of kidney epithelial cells. However, the T22N mutant of this GTPase, which acts as a dominantnegative variant, was excluded from the ciliary compartment. Coexpression of this mutant together with SmoA1.Venus and Kim1.Venus caused substantial reduction of the trafficking rate for both ciliary proteins. At the same time, expression of the endocytic GTPase Rab5, locked in an inactive state (S34N), specifically decreased a speed of Kim1.Venus delivery into the cilium but not the ciliary transport of Smoothened. These data provide novel insights into the regulation of transport into the primary cilium by Rab GTPases.
Author: Bashkurov, Mikhail
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7036731256