| Description |
Metazoan cells undergo an open mitosis during which interphase nuclear architecture is dismantled. Once chromosomes are separated by the mitotic spindle during anaphase, membrane and associated nuclear envelope proteins begin to target to the chromatin surface. As the nascent nuclear envelope engulfs chromatin disks, certain transmembrane proteins form discrete subdomains associated with different processes: nuclear pore complexes rapidly assemble at the peripheral edge of chromatin disks while cellular machinery facilitates microtubule clearance and membrane sealing at central regions. These initial targeting events are expeditious but transient, occurring on the order of minutes, and are then lost as the nuclear envelope seals and establishes a permeability barrier. As cells transition to telophase, sealing machinery is cleared from the chromatin surface, subdomains redistribute, and new nuclear pore complexes must be inserted into the double membrane of the nuclear envelope. The endosomal sorting complex required for transport (ESCRT) pathway and associated AAA-ATPases clear microtubules from the chromatin surface and seal the nascent nuclear envelope. In this dissertation, I report that the inner nuclear membrane protein LEM2 interacts directly with the ESCRT factor CHMP7 to recruit additional ESCRT factors at the nascent nuclear envelope during late anaphase. I show that depleting LEM2 abrogates CHMP7 - and downstream ESCRT-III - localization in anaphase by both fixed and live cell imaging. iv Additionally, these studies show that disrupting the function of the nuclear pore protein Nup153 interferes with ongoing addition of specific constituents to the nascent nuclear envelope after anaphase. This phenotype develops early in telophase, after the nuclear envelope initially encloses chromatin, revealing late events of nuclear formation in mammalian cells. Further, protein targeting defects are restricted to a subset of inner nuclear membrane proteins, pointing to differential requirements for ongoing targeting. Disrupting Nup153 function in nuclear formation reveals that two phases of nuclear membrane addition can be uncoupled in mammalian cells and that there is unanticipated complexity in targeting residents of the nuclear envelope after mitosis. |
