Abstract
In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle. However, how the NE and NPC reassemble remains largely unclear. Here, using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly. We observed that the AL are parallel membrane-pair stacks and possess regularly spaced AL pore complexes (ALPCs) that are morphologically similar to the NPCs. We found that the AL assemble in the cytoplasm during mitotic exit simultaneously with NE re-formation in daughter cells. Then, the assembled AL either bound the decondensing chromatin to directly transform into the NE or bound and fused with the outer nuclear membrane to join the assembling NE. The AL did not colocalize with sheet and tubular endoplasmic reticulum (ER) marker proteins on the ER or the lamin B receptor-localized membrane in the cytoplasm, suggesting that postmitotic AL assembly occurs independently of the chromatin and ER. Collectively, our results indicate that postmitotic AL assembly is a common cellular event and an intermediate step in NE and NPC assembly and in NE expansion in higher eukaryotic cells.
Highlights
In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle
Using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly
We demonstrate that both the NE and AL assemble simultaneously at the end of mitosis and that postmitotic AL assembly contributes to NE assembly and expansion by serving as an intermediate step in NE reconstitution
Summary
To deeply investigate the mechanism of NE and NPC assembly, we reexamined nuclear assembly processes by tracing dynamic localizations of the NE protein lamin B receptor (LBR), the ER protein calnexin, and Nup proteins in HeLa cells (Fig. 1, A and B). We found that the GFP– Nups incorporated into the NPCs in interphase and dispersed into the cytoplasm in mitosis (Fig. 2A, cell 1 (C1) and cell 2 (C2); Fig. S2; Movies S1 and S2) When these cells divided, the GFP–Nup-containing particles simultaneously assembled near the separated chromatids and gradually assembled into the assembling NE (Fig. 2A). Using structural illumination microscopy for super-resolution livecell imaging, we observed that some of the AL contacted the ER, which was consistent with a previous report [29], whereas others did not (Fig. S4D) Taken together, these results demonstrate that in addition to the ER and previously disassembled NE membrane vesicles, the membrane and ALPCs of the AL are sources of the NE and NPCs and that AL assembly and ER reconstruction are relatively independent of each other
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