The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Drosophila Kelch Is an Oligomeric Ring Canal Actin Organizer

Drosophila kelch has four protein domains, two of which are found in kelch-family proteins and in numerous nonkelch proteins. In Drosophila, kelch is required to maintain ring canal organization during oogenesis. We have performed a structure–function analysis to study the function of Drosophila kelch. The amino-terminal region (NTR) regulates the timing of kelch localization to the ring canals. Without the NTR, the protein localizes precociously and destabilizes the ring canals and the germ cell membranes, leading to dominant sterility. The amino half of the protein including the BTB domain mediates dimerization. Oligomerization through the amino half of kelch might allow cross-linking of ring canal actin filaments, organizing the inner rim cytoskeleton. The kelch repeat domain is necessary and sufficient for ring canal localization and likely mediates an additional interaction, possibly with actin.     

Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.     

An InCytes from MBC Selection: Murine CENP-F Regulates Centrosomal Microtubule Nucleation and Interacts with Hook2 at the Centrosome

The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F^−/− cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F^−/− cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.     

Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, it was found that null mutations in the dCTCF gene died as pharate adults, which failed to eclose from their pupal case, or shortly after hatching of adults. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears is limited mainly to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not important for the functional activity of dCTCF in vivo.     

A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

The kinesin superfamily is a large group of proteins (kinesin-like proteins [KLPs]) that share sequence similarity with the microtubule (MT) motor kinesin. Several members of this superfamily have been implicated in various stages of mitosis and meiosis. Here we report our studies on KLP67A of Drosophila. DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain. To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction. We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types. In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern. Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled. These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.     

The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin

AKAP450 (also known as AKAP350, CG-NAP or Hyperion) and pericentrin are large coiled-coil proteins found in mammalian centrosomes that serve to recruit structural and regulatory components including dynein and protein kinase A. We find that these proteins share a well conserved 90 amino acid domain near their C-termini that is also found in coiled-coil proteins of unknown function from Drosophila and fission yeast. Fusion of the C-terminal region from either protein to a reporter protein confers a centrosomal localization, and overexpression of the domain from AKAP450 displaces endogenous pericentrin, suggesting recruitment to a shared site. When isolated from transfected cells the C-terminal domain of AKAP450 was associated with calmodulin, suggesting that this protein could contribute to centrosome assembly.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.     

Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation

Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus end–binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1’s MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high-MT-affinity TgEB1 mutant promotes the formation of overstabilized MT bundles, resulting in avulsion of otherwise tightly clustered kinetochores. Overall we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.     

Csi1p recruits alp7p/TACC to the spindle pole bodies for bipolar spindle formation

Accurate chromosome segregation requires timely bipolar spindle formation during mitosis. The transforming acidic coiled-coil (TACC) family proteins and the ch-TOG family proteins are key players in bipolar spindle formation. They form a complex to stabilize spindle microtubules, mainly dependent on their localization to the centrosome (the spindle pole body [SPB] in yeast). The molecular mechanism underlying the targeting of the TACC–ch-TOG complex to the centrosome remains unclear. Here we show that the fission yeast Schizosaccharomyces pombe TACC orthologue alp7p is recruited to the SPB by csi1p. The csi1p-interacting region lies within the conserved TACC domain of alp7p, and the carboxyl-terminal domain of csi1p is responsible for interacting with alp7p. Compromised interaction between csi1p and alp7p impairs the localization of alp7p to the SPB during mitosis, thus delaying bipolar spindle formation and leading to anaphase B lagging chromosomes. Hence our study establishes that csi1p serves as a linking molecule tethering spindle-stabilizing factors to the SPB for promoting bipolar spindle assembly.     

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.     

CEP215 is involved in the dynein-dependent accumulation of pericentriolar matrix proteins for spindle pole formation

CEP215 is a human orthologue of Drosophila centrosomin which is a core centrosome component for the pericentriolar matrix protein recruitment. Recent investigations revealed that CEP215 is required for centrosome cohesion, centrosomal attachment of the g-TuRC, and microtubule dynamics. However, it remains to be obscure how CEP215 functions for recruitment of the centrosomal proteins during the centrosome cycle. Here, we investigated a role of CEP215 during mitosis. Knockdown of CEP215 resulted in characteristic mitotic phenotypes, including monopolar spindle formation, a decrease in distance between the spindle pole pair, and detachment of the centrosomes from the spindle poles. We noticed that CEP215 is critical for centrosomal localization of dynein throughout the cell cycle. As a consequence, the selective centrosomal proteins were not recruited to the centrosome properly. Finally, the centrosomal localization of CEP215 also depends on the dynein-dynactin complex. Based on the results, we propose that CEP215 regulates a dynein-dependent transport of the pericentriolar matrix proteins during the centrosome maturation.     

EAST affects the activity of Su(Hw) insulators by two different mechanisms in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.     

Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change

FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (∼ca. 70 Å) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.     

A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity

The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity in vitro and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase in vivo as well as in vitro. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.     

Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly

Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.     

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array

XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization.