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Mitotic spindle formation into a bipolar structure suitable for chromosome segregation requires reorganization of the interphase microtubule cytoskeleton. A multi-protein complex at spindle poles acts as a microtubule organizing center (MTOC). Microtubules are assembled from this site through the addition of α/β-tubulin heterodimers onto a template complex containing γ-tubulin (γ-TuSC) imbedded into a larger macromolecular ring (γ-TuRC). Our goal is to apply insights on patterning biological polymers in vivo to development of hybrid biosynthetic systems capable of utilizing microtubules in self-assembling metamorphic patterns including parquet deformation behavior. Dynamic patterning has applications in biosensing, materials design and new nanomanufacturing paradigms. Building off of recent structural insights into γ-TuSC and GCP4 with our own detailed genetic analysis, site-directed mutagenesis, cross-species functional studies and biochemical purification and nucleation assays we provide novel insights on MTOC structural requirements to nucleation. Additionally we have identified an associated regulatory mechanism utilized by a subset of Kinesin-14 members for targeting and regulatory interference at poles (TRIP) distinct from microtubule targeting elements found in other Kinesin-14 members such as Drosophila Ncd. Our in vivo analysis supports application of the recent Kollman-Agard structure as a general eukaryotic model however with species-specific protein and domain constraints as well as contact sites for Kinesin-14 regulation of γ-TuRC. Our findings have broad application towards a general understanding of cellular MTOC machinery and reiterates the flexibility of Klps to localize to multiple spindle compartments and participate in a diversity of cellular roles. Natural cellular machines provide tools and inspiration for biomimicry and for resolution of societal grand challenges through redirected applications. Using tools available through the semiconductor industry and materials science we are generating artificial centrosomes (ACENs) from nanoparticles to act as simplified nucleation centers for use in synthetic self-assembly paradigms.
Biophysics
Biophysics
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