Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

Abstract

The nucleoporin Nup153 is a multifunctional protein and the mitotic checkpoint protein Mad1one of its many binding partners. The functional relevance of their interaction has remained elusive. Here, we have further dissected Nup153’s and Mad1’s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the Nup153-Mad1 interaction. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of the membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between the inner and outer nuclear membranes. Nup153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.SummaryThe mitotic checkpoint protein is required for Nup153 recruitment to anaphase chromatin and in turn post-mitotic, but not interphase nuclear pore complex assembly.

Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase

“…Although strong kinetochore association of Bub1-Bub3 complex was noticed in cdc15-2 fin1Δ mutant cells in anaphase, no Mad1 was detected at the anaphase kinetochore in cdc15-2 fin1Δ or asynchronous fin1Δ anaphase cells ( S2 Fig ). In these cells, Mad1 showed a characteristic nuclear envelop localization as described [ 28 ]. Thus, a distinct mechanism controls Mad1 kinetochore dissociation, which is likely the key to SAC silencing.…”