Structure and Mechanism: Hsp90 proteostasis, cilia biogenesis and the jumbo phage “nucleus”

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ABSTRACT My previous MIRA period focused on mechanisms of microtubule nucleation, centrosome structure and the phage-encoded cytoskeleton and “nucleus”. Now that my HHMI has ended, our strong efforts on protein ho- meostasis are included in this MIRA proposal. Throughout, our work seeks to understand fundamental molecu- lar mechanisms that underly cellular function. Where possible, complex systems are reconstituted in vitro and analyzed in atomic detail with the implications explored at a cellular level. The research has three parts. I. Birth, life and destruction: mechanisms of Hsp90/Hsp70-driven proteostasis: Maintenance of the cellular proteome is one of the most fundamental aspects of all organisms. Molecular chaperones facilitate folding and activation, sequester or recover aggregated proteins, participate in the removal of irreversibly mis- folded proteins, and help regulate folding capacity according to cellular need. While critical players have been identified, the molecular mechanisms by which most of these tasks are accomplished remain unknown. We focus on the cytosolic Hsp90 chaperones that facilitate the folding and activation of ~10% of the proteome. Hsp90's “clients” are enriched in proteins important for cellular signaling, proliferation, and survival making Hsp90 a valuable therapeutic target for multiple diseases. Despite the biological importance, the underlying mechanism of client remodeling is unknown, as is how the chaperones facilitate folding vs degradation triage decisions by presenting clients to E3 ligases. Through in vitro reconstitution, extensive biochemical, biophysi- cal and cryoEM structural analyses our goal is to elucidate the molecular mechanisms of these processes. II. Structure of the basal body transition zone, tomography technology: In non-dividing cells, centrioles mature into basal bodies that dock at the membrane leading to the formation of a primary cilium which serves as a sensory organelle on virtually all animal cells, or motile cilia to move fluid. These structures are important in numerous human diseases, including cancer and a broad array of ciliopathies. Unfortunately, there is only limited understanding of centriole or basal body structure, how the basal body docks at the membrane, transi- tions to an axoneme, or provides a distinct cellular compartment. We will use cultured mouse tracheal epithelial cells which can be grown and differentiated on grids to produce arrays of motile cilia. Cells will be high pres- sure frozen and FIB-milled to create thin lamella for high-resolution in situ cryoEM. Importantly, key proteins can be knocked out by CRISPR or tagged with Ferri-tag for simultaneous like/cryoEM visualization. Phage “nucleus” and host immunity evasion: The cell biology being revealed by Phi-KZ jumbo phages is simply extraordinary (collaboration Pogliano, UCSD), demonstrating what appears to be an entirely new concept in compartment formation. Upon infection, these phage form a “nucleus” from a self-assembling pro- tein monolayer shell that is centered by a dynamically unstable tubulin cytoskeleton. The shell grows as the phage DNA replicates, selectively imports DNA replication and transcription machinery, yet excludes cytosolic proteins and GFP. Collaborating with (Bondy-Denomy, UCSF) has shown that the shell confers resistance to all known host immunity factors (CRISPRs, restriction endoncleases). The molecular basis for these processes is unknown. We focus on the determining shell assembly principles and the mechanism of selective transport.