Regulation of Cellular Asymmetry During Mitosis

The RanGTP gradient is essential for nuclear transport, spindle organization and nuclear envelope formation. RCC1 is the only known exchange factor for Ran. The association of RCC1 with chromatin is crucial for generation of the Ran gradient, and defects in binding cause chromosome mis-segregation. RCC1 binding to chromatin is enhanced by apo-Ran. However, the mechanism is unclear. We now demonstrate that the N-terminal tail of RCC1 is essential for association with DNA but inhibits histone binding. Apo-Ran significantly promotes RCC1 binding to both DNA and histones, and these effects are tail-mediated. Using a FRET biosensor, we detect conformational changes in the tail of RCC1 coupled to the two binding modes and in response to interactions with Ran and importin-. The biosensor also reports changes accompanying mitosis in living cells. We propose that Ran induces a conformational switch in the tail that exposes the histonebinding surface on RCC1 and facilitates association of the positively-charged tail with DNA. Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. Confinement of the Pins protein to the lateral cortex is essential for this process. When epithelial cysts are grown in 3D cultures, spindle misorientation creates multiple lumens. We now show that silencing of Par3 causes spindle misorientation in MDCK cysts. Depletion of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically-tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized over the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC phosphorylates Pins on Ser401, which recruits 14-3-3 proteins. 14-3-3 binding inhibits association of Pins with Gi, through which Pins attaches to the cortex, so Pins falls off the apical cortex. Pins(S401A) mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. We iiconclude that the Par3 and aPKC polarity proteins ensure correct spindle orientation by excluding Pins from the apical cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation. iiiDedication This dissertation is dedicated to my grandfather Zheng Yue, my parents Weiyuan Hao & Tingfang Yue, my husband Shengcheng Han and our daughters Yu (Emily) Han & Lu (Caroline) Han for their deep love and constant support. This dissertation is dedicated to the Heavenly Father and the Lord Jesus Christ for His great Love, Mercy and Grace upon me. Without His mercy, this dissertation would never have been possible.

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