Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad1-Dependent Anaphase Inhibitor

Abstract

Summary The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bi-oriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the intramitotic production of a diffusible APC/CCdc20 inhibitor. Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit “wait anaphase” signals that preserve genome integrity.

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

“…Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1,2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4][5][6][7][8][9].…”

“…When both steps are abrogated, cells neither initiate nor maintain the SAC and thus exit mitosis with kinetics set by nuclear pore-derived MCC [3].…”

“…This contribution was revealed when cells bearing an NPC-defective Mad1 allele were treated with Aurora B inhibitors, which delay kinetochore recruitment of Mad1 relative to NEBD [31,32]. Under these conditions, cells entered mitosis without premade MCC and could not generate it quickly enough at kinetochores to prevent mitotic exit [3]. Because RZZ-deficient cells initiate but do not maintain the SAC, we anticipated a similar synthetic interaction with Aurora B inhibition.…”

“…NPC-bound Mad1-Mad2 generates MCC during interphase, thereby increasing the SAC's sensitivity and robustness [3]. This contribution was revealed when cells bearing an NPC-defective Mad1 allele were treated with Aurora B inhibitors, which delay kinetochore recruitment of Mad1 relative to NEBD [31,32].…”

“…The first occurs at NEBD, when Mad1-Mad2 released from nuclear pores is targeted to newly assembled kinetochores by Aurora B [3,31,43]. In the second step, Mad1-Mad2 becomes stably tethered to kinetochores by RZZ, extending SAC surveillance and mitotic arrest more than 10-fold.When both steps are abrogated, cells neither initiate nor maintain the SAC and thus exit mitosis with kinetics set by nuclear pore-derived MCC [3].Compelling evidence indicates that Bub1 is the kinetochore receptor for Mad1-Mad2 in yeast and nematodes [44,45]. In contrast, studies in mammalian cells differ as to (1) whether Bub1 recruits Mad1-Mad2 to kinetochores and (2) whether and how Bub1 contributes to SAC arrest [4-7, 9, 41].…”