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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2023)

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.

    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2022)
    The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1-positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.
      Creative Commons 'BY-NC' version 4.0 license
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2024)
      The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.
        Cover page: Kinetic investigation reveals an HIV-1 Nef-dependent increase in AP-2 recruitment and productivity at endocytic sites
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2019)
          The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nef's ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.
            Cover page: How HIV Nef Proteins Hijack Membrane Traffic To Promote Infection
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2024)
            Clathrin-mediated endocytosis (CME) is a critical trafficking process that begins when an elaborate endocytic protein network is established at the plasma membrane. Interaction of early endocytic proteins with anionic phospholipids and/or cargo has been suggested to trigger CME initiation. However, the exact mechanism by which CME sites are initiated has not been fully elucidated. In the budding yeast Saccharomyces cerevisiae, higher levels of anionic phospholipids and cargo molecules exist in the newly formed daughter cell compared to the levels in the mother cell during polarized growth. Taking advantage of this asymmetry, we quantitatively compared CME proteins in S. cerevisiae mother versus daughter cells, observing differences in the dynamics and composition of key endocytic proteins. Our results show that CME site initiation occurs preferentially on regions of the plasma membrane with a relatively higher density of endocytic cargo and/or acidic phospholipids. Furthermore, our combined live cell-imaging and yeast genetics analysis provided evidence for a molecular mechanism in which CME sites are initiated when Yap1801 and Yap1802 (yeast CALM/AP180) and Syp1 (yeast FCHo1/2) coordinate with anionic phospholipids and cargo molecules to trigger Ede1 (yeast Eps15)-centric CME initiation complex assembly at the plasma membrane.
              Cover page: The conserved protein adaptors CALM/AP180 and FCHo1/2 cooperatively recruit Eps15 to promote the initiation of clathrin-mediated endocytosis in yeast.
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