Nucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153–Mad1 association. 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 membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

核孔蛋白 Nup153 是一种多功能蛋白，也是有丝分裂检查点蛋白 Mad1（也称为 MAD1L1）的已知结合伴侣。它们相互作用的功能相关性仍然难以捉摸。在这里，我们进一步剖析了 Nup153 和 Mad1 的接口和功能相互作用。使用原位邻近连接测定，我们发现核膜 (NE) 的存在是 Nup153-Mad1 关联的先决条件。延时显微镜显示，Mad1 的缺失会延迟 Nup153 向后期染色质的募集，这通常伴随着后期的延长。此外，通过电子显微镜和三维结构照明研究发现，Nup153 和 Mad1 的耗尽导致了 NE 结构的改变，其特征是核孔复合体 (NPC) 膜曲率的变化以及内部和外部之间的间距扩大。核膜。Nup153 缺失（而非 Mad1 缺失）会导致间期 NPC 组装缺陷，导致细胞质核孔蛋白部分移位和 NPC 密度降低。综上所述，我们的结果表明，Nup153 在 NE 和 NPC 形成中具有不同的作用：在有丝分裂后 NE 重组中与 Mad1 协同作用，在间期 NPC 组装中独立于 Mad1。