The nuclear permeability barrier depends on closure of nuclear envelope (NE) holes. Here, we investigate closure of the NE opening surrounding the meiotic spindle in C. elegans oocytes. ESCRT-III components accumulate at the opening but are not required for nuclear closure on their own. 3D analysis revealed cytoplasmic membranes directly adjacent to NE holes containing meiotic spindle microtubules. We demonstrate that the NE protein phosphatase, CNEP-1/CTDNEP1, controls de novo glycerolipid synthesis through lipin to prevent invasion of excess ER membranes into NE holes and a defective NE permeability barrier. Loss of NE adaptors for ESCRT-III exacerbates ER invasion and nuclear permeability defects in cnep-1 mutants, suggesting that ESCRTs restrict excess ER membranes during NE closure. Restoring glycerolipid synthesis in embryos deleted for CNEP-1 and ESCRT components rescued NE permeability defects. Thus, regulating the production and feeding of ER membranes into NE holes together with ESCRT-mediated remodeling is required for nuclear closure.
Transient nuclear envelope (NE) ruptures in the Caenorhabditis elegans zygote are caused by a weakened nuclear lamina during nuclear positioning. Dynein-pulling forces enhance the severity of ruptures, while lamin restricts nucleocytoplasmic mixing and allows stable NE repair. This work is the first mechanistic analysis of NE rupture and repair in an organism.
Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28’s conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.
mel-28 (maternal-effect-lethal-28) encodes a conserved protein required for nuclear envelope function and chromosome segregation in Caenorhabditis elegans. Because mel-28 is a strict maternal-effect lethal gene, its function is required in the early embryo but appears to be dispensable for larval development. We wanted to test the idea that mel-28 has postembryonic roles that are buffered by the contributions of other genes. To find genes that act coordinately with mel-28, we did an RNA interference−based genetic interaction screen using mel-28 and wild-type larvae. We screened 18,364 clones and identified 65 genes that cause sterility in mel-28 but not wild-type worms. Some of these genes encode components of the nuclear pore. In addition we identified genes involved in dynein and dynactin function, vesicle transport, and cell-matrix attachments. By screening mel-28 larvae we have bypassed the requirement for mel-28 in the embryo, uncovering pleiotropic functions for mel-28 later in development that are normally provided by other genes. This work contributes toward revealing the gene networks that underlie cellular processes and reveals roles for a maternal-effect lethal gene later in development.
The nuclear envelope (NE) assembles and grows from bilayer lipids produced at the endoplasmic reticulum (ER). How ER membrane incorporation coordinates with assembly of nuclear pore complexes (NPCs) to generate a functional NE is not well understood. Here, we use the stereotypical first division of the early C. elegans embryo to test the role of the membrane-associated nucleoporin Ndc1 in coupling NPC assembly to NE formation and growth. 3D-EM tomography of reforming and expanded NEs establishes that Ndc1 determines NPC density. Loss of ndc1 results in faster turnover of the outer scaffold nucleoporin Nup160 at the NE, providing an explanation for how Ndc1 controls NPC number. NE formation fails in the absence of both Ndc1 and the inner ring component Nup53, suggesting partially redundant roles in NPC assembly. Importantly, upregulation of membrane synthesis restored the slow rate of nuclear growth resulting from loss of ndc1 but not from loss of nup53. Thus, membrane biogenesis can be decoupled from Ndc1-mediated NPC assembly to promote nuclear growth. Together, our data suggest that Ndc1 functions in parallel with Nup53 and membrane biogenesis to control NPC density and nuclear size.
Nuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble to form a functional NE after open mitosis is not known. Recruitment of the outer ring Nup107-160 complex to the NE initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3-6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7-9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of NE reformation revealed a significant decrease in NPC density in ndc1 deleted embryos: continuous nuclear membranes contained few holes where NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. Nup160 is not recruited to the nuclear rim when both ndc1 and nup53 are absent and nuclear assembly fails. This suggests that Ndc1 and Nup53 function in part in parallel pathways to drive post-mitotic nuclear assembly in vivo. Together, we show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold with nascent NEs to facilitate NPC assembly after open mitosis, revealing a previously uncharacterized role for Ndc1 in forming functional NE.
The nuclear permeability barrier depends on closure of holes in the nuclear envelope (NE). Here, we use meiotic C. elegans oocytes to demonstrate that local control of glycerophospholipid synthesis by CNEP-1/CTDNEP1 regulates the insertion of ER sheets into NE holes and functions independently of ESCRT-III to ensure NE closure.Deletion of CNEP-1 causes excess incorporation of ER membranes into NE holes and a defective NE permeability barrier. ESCRT-III components accumulate at the NE opening surrounding the meiotic spindle, and loss of NE adaptors for ESCRT-III exacerbates NE sealing defects in cnep-1 mutants. Limiting ER sheet production by restoring glycerophospholipid synthesis in cnep-1 mutants rescued NE permeability defects. 3D analysis showed that membrane sheets feed into and narrow NE holes occluded by meiotic spindle microtubules supporting a role for ER sheet insertion in NE closure.Thus, feeding of ER sheets into NE holes must be coordinated with production of ER sheets near the NE to promote NE closure.
Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance to in vivo events is not known. Here, we use C. elegans embryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE ruptures in vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct phase in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE ruptures in vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.
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