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Reporting in this issue of Developmental Cell, Linder et al. (2017) and Martino et al. (2017) reveal in highly complementary studies that Plk1 is recruited to the nuclear pore complex upon mitotic entry, where it acts with Cdk1 to hyperphosphorylate nucleoporin interfaces to promote NPC disassembly and nuclear envelope breakdown.
Copyright © 2017 Elsevier Inc. All rights reserved.
Amyotrophic lateral sclerosis (ALS) is a lethal late onset motor neuron disease with underlying cellular defects in RNA metabolism. In prior studies, two deleterious heterozygous mutations in the gene encoding human (h)Gle1 were identified in ALS patients. hGle1 is an mRNA processing modulator that requires inositol hexakisphosphate (IP) binding for function. Interestingly, one hGLE1 mutation (c.1965-2A>C) results in a novel 88 amino acid C-terminal insertion, generating an altered protein. Like hGle1A, at steady state, the altered protein termed hGle1-IVS14-2A>C is absent from the nuclear envelope rim and localizes to the cytoplasm. hGle1A performs essential cytoplasmic functions in translation and stress granule regulation. Therefore, we speculated that the ALS disease pathology results from altered cellular pools of hGle1 and increased cytoplasmic hGle1 activity. GFP-hGle1-IVS14-2A>C localized to stress granules comparably to GFP-hGle1A, and rescued stress granule defects following siRNA-mediated hGle1 depletion. As described for hGle1A, overexpression of the hGle1-IVS14-2A>C protein also induced formation of larger SGs. Interestingly, hGle1A and the disease associated hGle1-IVS14-2A>C overexpression induced the formation of distinct cytoplasmic protein aggregates that appear similar to those found in neurodegenerative diseases. Strikingly, the ALS-linked hGle1-IVS14-2A>C protein also rescued mRNA export defects upon depletion of endogenous hGle1, acting in a potentially novel bi-functional manner. We conclude that the ALS-linked hGle1-c.1965-2A>C mutation generates a protein isoform capable of both hGle1A- and hGle1B-ascribed functions, and thereby uncoupled from normal mechanisms of hGle1 regulation.
Copyright © 2015 Elsevier Ltd. All rights reserved.
The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.
© 2015 Casey et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus-centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, although the mechanisms underlying this regulation remain unknown. We also identified ASUN as a functional component of Integrator (INT), a multisubunit complex required for 3'-end processing of small nuclear RNAs. We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE. Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN-small interfering RNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. In addition, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support a model in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.
Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.
The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother-daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs.
The nuclear envelope (NE) in eukaryotic cells serves as the physical barrier between the nucleus and cytoplasm. Until recently, mechanisms for establishing the composition of the inner nuclear membrane (INM) remained uncharted. Current findings uncover multiple pathways for trafficking of integral and peripheral INM proteins. A major route for INM protein transport occurs through the nuclear pore complexes (NPCs) with additional requirements for nuclear localization sequences, transport receptors, and Ran-GTP. Studies also reveal a putative NPC-independent vesicular pathway for NE trafficking. INM perturbations lead to changes in nuclear physiology highlighting the potential human disease impacts of continued NE discoveries.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.
The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO(7)-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO(7)-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.
Over the last decade, the nuclear envelope (NE) has emerged as a key component in the organization and function of the nuclear genome. As many as 100 different proteins are thought to specifically localize to this double membrane that separates the cytoplasm and the nucleoplasm of eukaryotic cells. Selective portals through the NE are formed at sites where the inner and outer nuclear membranes are fused, and the coincident assembly of approximately 30 proteins into nuclear pore complexes occurs. These nuclear pore complexes are essential for the control of nucleocytoplasmic exchange. Many of the NE and nuclear pore proteins are thought to play crucial roles in gene regulation and thus are increasingly linked to human diseases.