PhD Oral Exam - Nhat Phi Pham, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Cytokinesis is the physical separation of a dividing cell into two daughters. Multiple pathways regulate cytokinesis to spatiotemporally couple the segregation of chromosomes and the constriction of the contractile ring. RhoA, the master regulator of cytokinesis, is activated at the equatorial cortex by its activator Ect2. This interaction requires Ect2 interacting with the centralspindlin complex, although how the Ect2-centralspindlin complex is recruited to the membrane is unclear. After ingression, Ect2 signal at the midbody decreases, and Ect2 is presumably re-localized to the nucleus, but this has not been studied. It is also unclear whether decreasing Rhoa activity is required after ingression for abscission. In this work, we show that the polybasic cluster region (PBC) contains a nuclear localization signal (NLS) that binds to importins, that this NLS is required for cytokinesis. Mutating this NLS can abolish Ect2 recruitment at the membrane. We propose that importin-binding facilitates the recruitment of Ect2 at the cortex, potentially by favouring a conformation with higher affinity for lipids. We also show that the nuclear re-sequestration of Ect2 is required for the stability of the intercellular bridge and abscission. Mutating the central NLS, which mediates nuclear import, causes sustained RhoA activity at the intercellular bridge. We propose that nuclear sequestration may regulate other contractile proteins to promote midbody maturation and abscission. Finally, to identify other contractile proteins regulated by importins, we perform a TurboID assay and identify 10 potentially novel interactors of importin-b1 in mitosis, with potential roles in cytokinesis.