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Protein IqgC iz protista Dictyostelium discoideum dijeli svojstva proteina iz skupina IQGAP i RasGAP te regulira endocitozu deaktivacijom male GTPaze RasG. Cilj ovog rada je karakterizacija dodatne uloge proteina IqgC u regulaciji stanične adhezije. Testovima adhezije pokazala sam da stanice mutanta iqgCimaju defekt u pričvršćivanju za podlogu. Ekspresijom skraćenih varijanti proteina IqgC u stanicama iqgCutvrdila sam da je za normalnu adheziju nužna prisutnost obje domene proteina, domene RGCt i domene GAP. Korištenjem mikroskopije TIRF ustanovila sam da je protein IqgC lokaliziran u fokalnim adhezijskim strukturama, gdje ga navodi domena RGCt. Biokemijskim pokusima i metodom BiFC ustanovila sam da protein IqgC veže malu GTPazu RapA. Analizom kretanja stanica otkrila sam anomalije u nasumičnom kretanju i kemotaksiji stanica mutanta iqgC- , vjerojatno posredovane deregulacijom aktivnosti GTPaza RasG i RapA. Analizom lokalizacije i aktivnosti proteina IqgC u staničnim sojevima deficijentnim za razne proteine regulatore adhezije identificirala sam signalni put reguliran proteinom RapA unutar kojeg protein IqgC djeluje kao pozitivni regulator adhezije. IqgC protein from the protist Dictyostelium discoideum shares the properties of IQGAP and RasGAP proteins and regulates endocytosis by deactivating small GTPase RasG. The goal of this work is an investigation of an additional role of IqgC in the regulation of adhesion. Using adhesion assays, I showed that mutant iqgCcells attach poorly to the substratum. Rescue experiments with truncated variants of IqgC showed that both RGCt and GAP domains are necessary for normal adhesion. Using TIRF microscopy, I established that IqgC is localized in focal adhesion structures and that the RGCt domain is responsible for its proper localization. Biochemistry and BiFC experiments identified an interaction between IqgC and RapA. Migration assays detected anomalies in random motility and chemotaxis of iqgCcells, likely mediated by deregulation of RasG and RapA signaling. By testing the localization and activity of the IqgC protein in cell strains deficient for various adhesion regulators, I identified a signaling pathway regulated by RapA where IqgC acts as a positive regulator of cell adhesion. |
