Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis

Abstract

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.

ZC3HC1 Is a Novel Inherent Component of the Nuclear Basket, Resident in a State of Reciprocal Dependence with TPR

“…In contrast to the reincorporation of NPC components like NUP153, which starts in anaphase, reattachment of ZC3HC1 to the post-mitotic NPCs happened much later. In fact, we found it to occur essentially concurrent to that of TPR, already known to become bound to the NPC only late in telophase or early in G1 when nuclear protein import activity has been resumed (Figure 5A; see also, e.g., [50,101,102]).…”

Dephosphorylation in nuclear reassembly after mitosis

“…Depletion of B55 also delays the recruitment of other Nups examined, reinforcing the idea that PP2A-B55 plays an essential role in timely NPC reassembly, possibly by dephosphorylating multiple Nups ( Cundell et al, 2016 ). Nup153 is a chromatin-associated protein that is targeted to chromosomes in late anaphase ( Bodoor et al, 1999 ). This Nup can interact directly with PP1 through a PP1-docking motif ( Moorhead et al, 2008 ).…”

Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway

“…Step 2: The TNPO1-cargo complex is translocated into the nucleus through the nuclear pore, which is anchored to microtubules and microfilaments in the cytoplasm (52). Step 3: Once in the nucleus and upon RanGTP binding to TNPO1, the NUP153 cargo dissociates from TNPO1 and is incorporated as a component of the NPC basket structure, which is responsible for anchoring TPR (18,19), the last nucleoporin to be assembled on the NPC (53). The resulting mature NPC is then fully functional for nucleocytoplasmic transport and other NPC-dependent roles, such as anchoring chromatin.…”