The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In C. elegans, PCM assembly requires the coiled-coil protein SPD-5. Here we found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

␤-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. Here we show that ␤-catenin is a component of interphase centrosomes and that stabilization of ␤-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. We show that ␤-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, ␤-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, ␤-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of ␤-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify ␤-catenin as a component of the intercentrosomal linker and define a new function for ␤-catenin as a key regulator of mitotic centrosome separation.[Keywords: ␤-Catenin; centrosome; Nek2; mitosis; C-Nap1; Rootletin] Supplemental material is available at http://www.genesdev.org. it has been shown that ␤-catenin localizes to centrosomes in mitosis and has a role in establishing a bipolar spindle (Kaplan et al. 2004); however, the regulation and function for ␤-catenin at centrosomes in normal cells and how this function is perturbed in cancers are not understood.Centrosomes undergo a highly regulated duplication cycle in interphase cells so that at the onset of mitosis a cell has two centrosomes that can separate to establish a bipolar spindle (Hinchcliffe and Sluder 2001;Tsou and Stearns 2006). In general, mechanisms of centrosome cohesion and separation are not well understood. It is thought that in interphase the two centrosomes, each with a pair of centrioles, are held together by a dynamic physical linker composed of C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin (Bahe et al. 2005;Yang et al. 2006). In mitosis, activation of the NIMArelated centrosomal kinase Nek2A results in the phosphorylation of C-Nap1 and Rootletin, which is thought to cause their dissociation from centrosomes and enable centrosome separation (Fry et al. 1998a;Mayor et al. 2002

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by ϵ-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.

Schroeder et al. quantitatively evaluate ER architecture in live cells at a ∼50-nm resolution through stimulated emission depletion (STED) microscopy. The ER is not limited to uniform sheets and tubules; they observe dynamic, nanoscale-size holes in ER sheets termed “nanoholes,” and they characterize the effects of perturbations of reticulons, Climp63, and the microtubule cytoskeleton on ER membrane nanostructures.

Background: Lipins are phosphatidic acid phosphatases. In yeast, lipin is activated by the Nem1p-Spo7p complex. There is controversy as to whether a mammalian Spo7p ortholog exists. Results: The metazoan Spo7p ortholog is now identified and shown to interact with lipins in yeast, nematodes, and mammalian cells. Conclusion: NEP1-R1 is the metazoan Spo7p ortholog. Significance: The lipin activation system is conserved in evolution.

The nuclear envelope is a subdomain of the endoplasmic reticulum (ER). Here we characterize CNEP-1 (CTD [Cterminal domain] nuclear envelope phosphatase-1), a nuclear envelope-enriched activator of the ER-associated phosphatidic acid phosphatase lipin that promotes synthesis of major membrane phospholipids over phosphatidylinositol (PI). CNEP-1 inhibition led to ectopic ER sheets in the vicinity of the nucleus that encased the nuclear envelope and interfered with nuclear envelope breakdown (NEBD) during cell division. Reducing PI synthesis suppressed these phenotypes, indicating that CNEP-1 spatially regulates phospholipid flux, biasing it away from PI production in the vicinity of the nuclear envelope to prevent excess ER sheet formation and NEBD defects.Supplemental material is available for this article.Received September 11, 2013; revised version accepted December 13, 2013. The endoplasmic reticulum (ER) is composed of sheets and tubules bounded by a membrane bilayer that faces the cytoplasm on one side and an internal lumen on the other. The ER is partitioned into subdomains specialized for different functions. The nuclear envelope subdomain is a spherical sheet perforated by nuclear pores that encases the chromatin. The outer membrane and lumen of the nuclear envelope are directly contiguous with other ER structural elements, whereas passage of proteins to the inner nuclear membrane (INM) is gated by the nuclear pores (Hetzer 2010;English and Voeltz 2013). The chromatin-facing INM is associated with the nuclear lamina, a dense filament meshwork that provides structural support (Hetzer 2010;Gerace and Huber 2012).The ER also serves as a platform for de novo phospholipid synthesis (Fagone and Jackowski 2009;Lagace and Ridgway 2013). A central player is the conserved phosphatase lipin, which converts phosphatidic acid (PA) to diacylglycerol (DAG) Siniossoglou 2013). In metazoans, lipin is at a branching point in the phospholipid synthesis pathway; the major building blocks of membrane bilayers (phosphatidylcholine [PC] and phosphatidylethanolamine [PE]) are synthesized from the lipin product DAG, whereas phosphatidylinositol (PI) is synthesized from the lipin substrate PA Lagace and Ridgway 2013;Siniossoglou 2013). PI is converted to phosphoinositides (PIPs) when transported to other organelles that house specific PIP kinases and phosphatases (Balla 2013). Within the ER, PC and PE make up >70% of total phospholipid, whereas PI makes up <10% (van Meer et al. 2008). In metazoans, decreased lipin activity is predicted to shift phospholipid flux away from production of DAG and the major membrane phospholipids PC and PE toward the production of PI. This is in contrast to budding yeast, where a pathway that is not present in metazoans (the CDP-DAG pathway) (Supplemental Fig. S1A) converts PA to major membrane phospholipids (Carman and Han 2011;Siniossoglou 2013). Thus, lipin inhibition in budding yeast leads to an increase in all phospholipids and an abnormal expansion of the nuclear envelope that does not occu...

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.