| Decades of research has demonstrated that cytoskeletal networks are important for providing positional information and directed movement of vesicle carriers and membrane-bound organelles. However, these studies have been almost exclusively restricted to the microtubule and actin cytoskeletons. In this dissertation, I present the first direct molecular link establishing a role for intermediate filaments in membrane protein sorting and organelle positioning by analyzing a novel functional interaction between the adaptor AP-3 and intermediate filaments. AP-3 is a member of the heterotetrameric adaptor complex family that specifically regulates sorting of transmembrane proteins to lysosomas and lysosome-related organelles. Accordingly, I have observed that both AP-3 and intermediate filament genetic deficiencies result in several common phenotypes at the cellular level, which include defects in zinc uptake, endo-lysosomal acidification, and autophagosome formation. In addition, I also found that both deficiencies caused impaired sorting of lysosomal proteins. These defects in endocytic organeller function occurred concomitantly with selective changes in lysosomal positioning. However, the AP-3/intermediate filament relationship appears not only to have effects on membrane protein sorting, but also on the filaments. Perturbations of adaptor dynamics using the drug Brefeldin A led to significant changes in the architecture of vimentin networks. This suggests that AP-3 and intermediate filaments likely have a reciprocal functional relationship, in which each component regulates the structure and/or function of the other. |
