Fluorescence Imaging of Virus-Host Cell Interaction and Super-Resolution Imaging of Neuronal Cytoskeleton

To study biological molecules, pathways and processes, fluorescence microscope has become an indispensable tool in modern biology. The major advantages of using fluorescence microscope include its ability to achieve molecule-specific visualization for targets of interest, and the compatibility of live cell imaging. With the advent of super-resolution imaging techniques, fluorescent microscope, for the first time, allows researchers to study biological processes on the nanometer scale. In this dissertation, I present the application of fluorescence imaging to characterize the role of host factors in influenza virus infection, and super-resolution imaging to study the developmental mechanism and prevalence of a periodic membrane skeleton in neurons. In Chapter 2 and 3, my colleagues and I studied the role of CD81 and COPI complex, two host factors identified in large-scale screens, for influenza infection. We found that CD81 regulates two distinct steps during influenza infection cycle: virus uncoating during entry and virus budding during egress. Depleting CD81 led to a significant defect in viral uncoating and viral gene replication during entry, while during virus egress, CD81 depletion resulted in virions that failed to detach from the plasma membrane and a marked decrease in progeny virus production. For COPI complexes, we found that COPI plays a direct role in viral membrane protein expression and assembly post-viral entry. In Chapter 4 and 5, we used super-resolution imaging to study the developmental mechanism and prevalence of a newly discovered periodic membrane skeleton in axons, formed by actin, spectrin and associated molecules. We found that the periodic membrane skeleton is highly prevalent in different neuronal types. It forms early during neuronal development, and originates from regions closer to the cell body and propagates toward the distal ends of axons. The lattice structure further matures by recruiting additional molecular components and appears to be highly stable once formed. The local concentration of βII spectrin is a key determinant for the formation of this periodic membrane skeleton. In addition, we identified ankyrin B as a critical molecular component for the polarized distribution of βII spectrin in neurites.Biology, Molecular and Cellula