UC Berkeley

The membrane trafficking pathway enables the specialization of cellular compartments by delivering proteins to specific destinations. This complex process is regulated by highly dynamic cellular factors whose spatiotemporal kinetics can be effectively studied through live cell microscopy. However, molecular mechanisms governing membrane trafficking is far from complete characterization due to the heterogeneous and interconnected nature of trafficking pathways within a cell.Here I describe studies focused on two methods to investigate membrane trafficking. The first study focuses on Nef, a viral accessory protein expressed by primate lentiviruses, and its interactions with the host endocytic and trans-Golgi secretory trafficking pathways. By focusing on the viral protein Nef and the endocytic or trans-Golgi trafficking associated proteins, I was able to isolate and investigate how the viral protein hijacks cellular traffic to promote infection. Through visualization of Nef interactions with endocytic protein AP-2, we determine how the productivity of endocytic pathways may be modulated by promoting the recruitment of AP-2. Furthermore, I found that AP-1, an adaptor protein associated with delivery of vesicles from the trans-Golgi network, is capable of forming tubulated compartments under clathrin knockdown conditions, that may serve a role in sequestering host MHC-I protein during HIV-1 infection. The second study explores fusion of genome-edited cells as a strategy for expediting the lengthy process of generating multi-colored cell lines through repeated genome-editing for microscopy studies. This approach will enable rapid screening of novel labeled protein combinations while avoiding artifacts such as aberrant morphology and dynamics, which are common consequences of overexpression, especially in the context of membrane trafficking. The dynamics observed in the fused cell lines more closely reflect the dynamics of endocytic proteins in a traditionally edited, multi-colored cell line when compared to cells that overexpress the same proteins. Finally, we utilize this method to screen protein-organelle interactions by fusing a query cell line with a collection of genome-edited cell lines expressing organelle markers.