A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labelled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

核孔复合物（NPC）的生物发生，是蛋白质自组装过程中一个依旧充满未解之谜的典型范例。我们新近开发了数种具有交叉反应性的抗核孔蛋白（nucleoporin, Nup）纳米抗体，可实现从蛙类到人类的完整核孔复合物成像。我们同时报道了一种简化的检测方法，通过添加荧光标记的抗核孔蛋白纳米抗体，可直接追踪有丝分裂后核孔复合物的组装过程。在细胞分裂间期，核孔复合物会嵌入已存在的核被膜中，但由于新组装的核孔复合物与已存在的复合物无法区分，实时监测这一过程极具挑战。我们通过将爪蟾来源的核孔复合物嵌入人类核被膜，并使用蛙类特异性抗核孔蛋白纳米抗体进行检测，攻克了这一难题。我们进一步探究了抗核孔蛋白纳米抗体是否可作为核孔复合物组装的抑制剂。通过针对保守表位的筛选策略，我们获得了可阻断核孔蛋白间相互作用界面并阻滞核孔复合物组装的抗Nup93、Nup98及Nup155纳米抗体。我们解析了纳米抗体-靶标复合物的结构，并明确了Nup93的α螺线管结构域在招募Nup358与Nup214·88·62复合物过程中的作用，同时阐明了Nup155及Nup98自酶解结构域在核孔复合物支架组装中的功能。这一发现提示存在一类检验点，将孔道形成与以Nup98为主导的通透性屏障组装过程相偶联。