Title: K-Fiber Dynamical Response to Molecular and Mechanical Perturbations
Speaker: Marc Begley

Abstract: The spindle segregates chromosomes during mitosis. Robust spindle function helps maintain cellular genetic integrity, as malfunction in chromosome segregation can lead to cancer or birth defects. Crucial to this machine are k-fibers, microtubule bundles that link chromosomes to the spindle poles. Electron and fluorescence microscopy studies have provided some insight into k-fiber structure, but the composition, architecture, and mechanics of k-fibers are largely unresolved. To probe k-fiber structure and dynamics, we mechanically perturb k-fibers in mammalian PtK2 cells by targeted laser ablation. For each ablation, we track the translation and occasional splaying of the resultant stub. We then characterize the relationships between these properties of stub dynamics to each other, as well as any effects changes to the concentration or functionality of select candidate inter-microtubule bridges might have on stub movement or splaying. Control data demonstrate that the interconnectivity of k-fiber microtubules is sometimes, but not always, strong enough to maintain the fiber’s structure following ablation, as some stubs splay while others do not. Although the structures and dynamical response of molecularly perturbed spindles agree with the recent findings of other labs, molecular perturbation experiments have yet to identify any frequent inter-MT bridges localizing specifically to k-fibers. We have, however, observed puzzling post-ablation stub behavior in many of these perturbed spindles, which we plan to further investigate. Additionally, we plan to perform the same ablation experiments in spindles subject to other molecular perturbations, as well as spindles of another mammalian species.
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Title: Laser ablation to probe furrow ingression mechanisms in Schizosaccharomyces pombe
Speaker: Mohamed Moshtohry

Abstract: Cytokinesis is essential for cell proliferation and differentiation. Failure of cytokinesis can lead to tetraploidy and aneuploidy. Cytokinesis involves the constriction of a contractile ring of actin and myosin that causes furrow ingression, which partitions the cytoplasm into two daughter cells. The mechanisms governing furrow ingression remain not well understood due to poor temporal and spatial resolution of those events, as well as the overlap of signaling pathways involved. Here, we combine laser microsurgery, quantitative fluorescence microscopy, hsFPALM and genetics to understand how the molecular organization of cytokinesis proteins leads to force production and furrow ingression. Going forward, we will be determining cytokinesis protein organization and quantifying the recoil kinetics following laser ablation before and after altering the number and molecular composition of the nodes of the cytokinesis proteins. Quantifying the mechanics of constricting rings will help us build a new model of contractility that further elucidates the mechanism of furrow ingression and reveals how the molecular organization of the contractile ring governs contractile force in live cells.