Abstract
The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.
Highlights
The nuclear pore complex (NPC) is the largest non-polymeric protein complex in eukaryotic cells and composed of multiple copies of around 30 different proteins termed nucleoporins (Nups) 1
A major reason for this gap in our knowledge was that individual NPCs and ER topology are below the resolution of live cell fluorescence microscopy that is needed to capture the dynamic process of postmitotic nuclear assembly, precluding reliable and quantitative observation of NPC
To measure how nuclear membrane sealing around DNA relates to NPC formation in space and time, we reconstructed whole dividing human cells with a time resolution of approximately one minute after the beginning of mitotic chromosome segregation by correlating single cell live imaging with focused ion beam scanning electron microscopy (FIB-SEM) (Fig. 1a and Supplementary Fig. 1)
Summary
The nuclear pore complex (NPC) is the largest non-polymeric protein complex in eukaryotic cells and composed of multiple copies of around 30 different proteins termed nucleoporins (Nups) 1. The process of postmitotic assembly of the NPC and the nuclear membranes from mitotic. How thousands of NPCs can assemble within a few minutes without interfering with the rapid sealing of NEs during mitotic exit has remained mysterious. A major reason for this gap in our knowledge was that individual NPCs and ER topology are below the resolution of live cell fluorescence microscopy that is needed to capture the dynamic process of postmitotic nuclear assembly, precluding reliable and quantitative observation of NPC assembly and the sealing of NE membranes. We combine live cell imaging with high resolution 3D electron microscopy to ultrastructurally reconstruct the dynamic process of postmitotic NPC and NE assembly
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