Correction: The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins.

[This corrects the article DOI: 10.1371/journal.pbio.1000281.].

Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells.

The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation.

The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation.

The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the γ-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.

CHD4 is a RanGTP-dependent MAP that stabilizes microtubules and regulates bipolar spindle formation.

BACKGROUND: Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS: We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS: Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.

Three-dimensional reconstruction of bacteria with a complex endomembrane system.

The division of cellular space into functionally distinct membrane-defined compartments has been one of the major transitions in the history of life. Such compartmentalization has been claimed to occur in members of the Planctomycetes, Verrucomicrobiae, and Chlamydiae bacterial superphylum. Here we have investigated the three-dimensional organization of the complex endomembrane system in the planctomycete bacteria Gemmata obscuriglobus. We reveal that the G. obscuriglobus cells are neither compartmentalized nor nucleated as none of the spaces created by the membrane invaginations are closed; instead, they are all interconnected. Thus, the membrane organization of G. obscuriglobus, and most likely all PVC members, is not different from, but an extension of, the "classical" Gram-negative bacterial membrane system. Our results have implications for our definition and understanding of bacterial cell organization, the genesis of complex structure, and the origin of the eukaryotic endomembrane system.

Mitotic lamin disassembly is triggered by lipid-mediated signaling.

Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis.

Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.

Samp1 is a component of TAN lines and is required for nuclear movement.

The position of the nucleus is regulated in different developmental stages and cellular events. During polarization, the nucleus moves away from the future leading edge and this movement is required for proper cell migration. Nuclear movement requires the LINC complex components nesprin-2G and SUN2, which form transmembrane actin-associated nuclear (TAN) lines at the nuclear envelope. Here we show that the nuclear envelope protein Samp1 (NET5) is involved in nuclear movement during fibroblast polarization and migration. Moreover, we demonstrate that Samp1 is a component of TAN lines that contain nesprin-2G and SUN2. Finally, Samp1 associates with SUN2 and lamin A/C, and the presence of Samp1 at the nuclear envelope requires lamin A/C. These results support a role for Samp1 in the association between the LINC complex and lamins during nuclear movement.

NLS-mediated NPC functions of the nucleoporin Pom121.

RanGTP mediates nuclear import and mitotic spindle assembly by dissociating import receptors from nuclear localization signal (NLS) bearing proteins. We investigated the interplay between import receptors and the transmembrane nucleoporin Pom121. We found that Pom121 interacts with importin alpha/beta and a group of nucleoporins in an NLS-dependent manner. In vivo, replacement of Pom121 with an NLS mutant version resulted in defective nuclear transport, induction of aberrant cytoplasmic membrane stacks and decreased cell viability. We propose that the NLS sites of Pom121 affect its function in NPC assembly both by influencing nucleoporin interactions and pore membrane structure.

The compartmentalized bacteria of the planctomycetes-verrucomicrobia-chlamydiae superphylum have membrane coat-like proteins.

The development of the endomembrane system was a major step in eukaryotic evolution. Membrane coats, which exhibit a unique arrangement of beta-propeller and alpha-helical repeat domains, play key roles in shaping eukaryotic membranes. Such proteins are likely to have been present in the ancestral eukaryote but cannot be detected in prokaryotes using sequence-only searches. We have used a structure-based detection protocol to search all proteomes for proteins with this domain architecture. Apart from the eukaryotes, we identified this protein architecture only in the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum, many members of which share a compartmentalized cell plan. We determined that one such protein is partly localized at the membranes of vesicles formed inside the cells in the planctomycete Gemmata obscuriglobus. Our results demonstrate similarities between bacterial and eukaryotic compartmentalization machinery, suggesting that the bacterial PVC superphylum contributed significantly to eukaryogenesis.

ISWI is a RanGTP-dependent MAP required for chromosome segregation.

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.

The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex.

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.

Lipin is required for efficient breakdown of the nuclear envelope in Caenorhabditis elegans.

The nuclear envelope (NE) is a double lipid bilayer that separates nucleus and cytoplasm. In metazoa, NE breakdown (NEBD) occurs during prophase and NE reformation around segregated chromatids occurs at anaphase-telophase. We identified Caenorhabditis elegans Lipin homologue (called Lpin-1) as an essential factor with roles in endoplasmic reticulum (ER) organization and NEBD. RNAi-mediated downregulation of Lpin-1 had no effect on timely entry into mitosis or on the early steps of NEBD, but Lpin-1 was required for disassembly of the nuclear lamina during late NEBD. This Lpin-1 requirement appears to be separable from the effect of Lpin-1 on the peripheral ER.

Hepatoma up-regulated protein is required for chromatin-induced microtubule assembly independently of TPX2.

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate.

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.