Regulating cytoskeleton-based vesicular transport within the early secretory pathway.

摘要

Efficient intracellular protein trafficking requires precise regulation or cytoskeleton-mediated motility. For example, the Golgi-localized GTP-hinding protein Cdc42 coordinates Golgi vesicle assembly with the recruitment of the motor protein dynein. Cdc42 in a complex with the COPI coat protein, coatomer, regulates both actin polymerization and dynein recruitment. In this thesis, this regulatory function of Cdc42 is confirmed by knock down of a Golgi specific Cdc42GAP: ARHGAP21. Cdc42-regulated dynein function is dependent on Arf1 activation since the Arf1 inhibitor brefeldin A blocked VSVGts045 dynein-dependent ER-to-Golgi trafficking. I also found that the conventional kinesin KIF5B localizes to Golgi membranes in NRK cells and, like dynein, binds to isolated Golgi membranes upon AFR1 activation. KIF5B appears to associate with coatomer-coated vesicular structures on the Golgi apparatus. More importantly, kinesin and dynein levels on COPI vesicles are regulated in a reciprocal manner. Disrupting the coatomer-Cdc42 complex with a peptide derived from the C-terminal tail of the cargo receptor p23 increases dynein levels while decreasing kinesin levels. Moreover, I characterized the roles of actin in Cdc42 regulated microtubule motor recruitment. Two related actin-binding proteins, mammalian Abp1 (mAbp1) and drebrin have different subcellular localization and bind to distinct actin structures on the Golgi apparatus in response to changing Cdc42 signaling. I used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. My results suggest specific actin-based structures are able to select specific actin-binding proteins and thus carry out multiple different functions within cells. My data indicate that the cargo-receptor-sensitive coatomer-Cdc42 complex helps select motor proteins during microtubule-dependent translocation of Golgi-derived vesicles through actin remodeling.