This component of the Program Project is a proposal to study structure and assembly of three intricate molecular machines ~ a non-enveloped virus, clathrin coats, and kinetochores. (1) A long-term goal of our work on non-enveloped viruses and on clathrin coats has been to connect high-resolution structure analysis (by x-ray crystallography and by single-particle cryoEM) with observations on the dynamics of individual particles by fluorescence imaging. CryoEM structures of rotavirus particles at near-atomic resolution and of clathrin coats at subnanometer resolution, determined in collaboration with members of the Program Project team, have led to experiments that define mechanisms of viral entry and clathrin assembly and uncoating, both in vitro and in living cells. We will now use cryoET to bridge between high-resolution structural analyses and live-cell imaging, in collaboration with Project 4. The objectives are to determine how a non-enveloped virus (rotavirus in the work here) perforates or disrupts the membrane of an intracellular vesicle in order to gain access to the cytosol ~ a critical and still puzzling step in the infectious process ~ and to visualize interactions of clathrin with other coat components. (2) Budding-yeast kinetochores assemble on centromeric DNA (-150 bp) and connect each chromosome to a single microtubule (MT) of the mitotic spindle. We have reconstituted the DNA-proximal "inner kinetochore" from recombinant proteins in vitro, and we have determined crystal structures of many of its components. We will obtain 3D reconstructions, from negatively stained preparations and by cryoEM where appropriate, of the reconstituted inner kinetochore and some of its subcomplexes, and build a molecular model of this -750 kDa protein:DNA assembly. From analysis of the inner-kinetochore structure and information on its interaction partners, we will add additional "linker" complexes, which connect the inner kinetochore with microtubule-attached "outer kinetochore" components, and determine further structures by EM.
Structural and mechanistic investigations of non-enveloped virus entry and clathrin-mediated uptake will enhance our understanding of how viruses, other pathogens, and toxins gain entry to a cell. Structural analysis of kinetochore architecture will yield fundamental new insights into the molecular basis of accurate chromosome segregation during cell division.
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