Spatiotemporal dynamics and roles of septins in late cytokinesis

Daniel M. Hwang

doi:10.17918/etd-7100

In the final event of cell division, the thin membrane bridge connecting dividing cells is severed in an event known as abscission. This completion of cell division must be coordinated with the segregation of chromatin into two distinct membrane compartments. When chromosome segregation fails to complete successfully, chromatin becomes trapped at the intracellular bridge and premature severing of this bridge can lead to changes in genetic material. In order to monitor the clearance of chromatin at the intracellular bridge, cells employ the NoCut checkpoint which acts through the Aurora B serine/threonine kinase. Leading up to abscission, two events characterize late cytokinesis: the compaction of microtubules at the intracellular bridge and the transition of spastin/ESCRTIII complex to the secondary ingression site where microtubules are disassembled and the membrane is severed. In this work, using immunofluorescence and live cell techniques, I have characterized the spatiotemporal dynamics of septins at the intracellular bridge with respect to microtubules as well as with spastin and ESCRTIII complex proteins. I have found that septins bind microtubules and form two membrane-associated rings. These two populations of septins display distinct spatiotemporal dynamics, suggesting separate roles in late cytokinesis. The microtubule binding population of septins appear to bundle microtubules and mutational analysis of serine residues, which matched the consensus sequence for Aurora B kinase phosphorylation, lead to changes in microtubule compaction, which were coincident with loss of septin association with microtubules at the intracellular bridge. Treatment with hesperadin, a small molecule inhibitor of Aurora B kinase also produced aberrant accumulation of septins at the intracellular bridge. Characterization of the spatiotemporal dynamics of septins with CHMP4B, CHMP2A and spastin showed that these proteins do not begin to accumulate strongly at the intracellular bridge or transition to the secondary ingression zone until septin rings disappear. I hypothesize that septins aid in the compaction of microtubules at the intracellular bridge and form diffusional barriers, which prevent the abscission machinery from transitioning to the abscission site until the NoCut checkpoint has been satisfied. Overall, I identified septins as potential components of the abscission machinery and regulators of abscission timing

Spatiotemporal dynamics and roles of septins in late cytokinesis

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Spatiotemporal dynamics and roles of septins in late cytokinesis

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