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Yixian Zheng

Publications and source records attributed to Yixian Zheng.

18 recordsLinked to original sources
PubMed2006

The mitotic spindle matrix: a fibro-membranous lamin connection.

The mitotic spindle apparatus has attracted the attention of cell biologists for decades. Whereas the main function of this microtubule-based system is to segregate chromosomes, spindle morphogenesis and chromosome segregation must also coordinate with the segregation of the whole cell. The finding that RanGTPase stimulates the assembly of a lamin B-containing membranous matrix in mitosis(1) may provide a connection between the segregation of mitotic chromosomes and the partitioning of membrane systems during cell division.

Chromosome Segregation↗
PubMed2006

Microtubule nucleation: gamma-tubulin and beyond.

Centrosomes and their fungal equivalents, spindle pole bodies (SPBs), are the main microtubule (MT)-organizing centers in eukaryotic cells. Several proteins have been implicated in microtubule formation by centrosomes and SPBs, including microtubule-minus-end-binding proteins and proteins that bind along the length or stabilize the plus ends of microtubules. Recent work has improved our understanding of the molecular mechanisms of MT formation. In particular, it has shown that gamma-tubulin and its associated proteins play key roles in microtubule nucleation and spindle assembly in evolutionarily distant species ranging from fungi to mammals. Other work indicates that gamma-tubulin-mediated microtubule nucleation, although necessary, is not sufficient for mitotic spindle assembly but requires additional proteins that regulate microtubule nucleation independently of centrosomes.

Animals↗
PubMed2006

A mitotic lamin B matrix induced by RanGTP required for spindle assembly.

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate-bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.

Animals↗
PubMed2005

Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly.

The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts . Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear . Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads , AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.

Animals↗
PubMed2005

Chromosome alignment and segregation regulated by ubiquitination of survivin.

Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. We report that a de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation.

Amino Acid Sequence↗
PubMed2004

Aurora kinases in spindle assembly and chromosome segregation.

The Aurora kinase family consists of three members, Aurora A, B, and C. The Aurora A and B kinases have emerged as essential regulators of cell division. While Aurora A kinase is implicated in regulating mitotic entry, centrosome maturation, and spindle assembly, Aurora B is required for correct chromosome segregation and cytokinesis. In this review, we summarize the roles of Aurora A and B in spindle assembly and chromosome segregation.

Animals↗
PubMed2004

The production and localization of GTP-bound ran in mitotic mammalian tissue culture cells.

The RanGTPase system has multiple functions in both interphase and mitosis. Extensive studies of Ran-driven nucleocytoplasmic transport have contributed significantly toward our understanding of how RanGTP is produced, hydrolyzed, and localized in interphase. However, there is still a lack of understanding about how this system operates in mitosis. Recent advances have begun to shed light on how RanGTP is produced and localized in mitotic mammalian cells.

Animals↗
PubMed2004

The Cdc48/p97-Ufd1-Npl4 complex: its potential role in coordinating cellular morphogenesis during the M-G1 transition.

The AAA ATPase Cdc48/p97 together with its adaptors, Ufd1-Npl4, regulate membrane-related functions and mitotic spindle disassembly by directly binding to membrane-associated proteins or spindle assembly factors, modulating their interactions with membranes or spindles, respectively. Here, we discuss the possibility that the Cdc48/ p97-Ufd1-Npl4 complex has a more general role in mediating morphological transitions as the cell exits mitosis and enters G(1).

Adenosine Triphosphatases↗
PubMed2004

Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells.

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. We show here that Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin alpha and beta to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells.

Animals↗
PubMed2004

G protein control of microtubule assembly.

Microtubules are dynamic polymers required for many aspects of eukaryotic cell function. The interphase microtubule network is essential for intracellular transport, organization, and cell polarization, whereas the mitotic spindle is required for chromosome segregation and cell division. Studies in different areas such as cell migration, mitosis, and asymmetric cell division have shown that Ran, Rho, and heterotrimeric G proteins regulate many aspects of microtubule functions. This review surveys how G protein-signaling coordinates microtubule polymerization and organization with specific cellular activities.

Animals↗
PubMed2003

The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis.

Spindle disassembly at the end of mitosis is a complex and poorly understood process. Here, we report that the AAA-ATPase Cdc48/p97 and its adapters Ufd1-Npl4, which have a well-established role in membrane functions, also regulate spindle disassembly by modulating microtubule dynamics and bundling at the end of mitosis. In the absence of p97-Ufd1-Npl4 function, microtubules in Xenopus egg extracts remain as monopolar spindles attached to condensed chromosomes after Cdc2 kinase activity has returned to the interphase level. Consequently, interphase microtubule arrays and nuclei are not established. Genetic analyses of Cdc48, the yeast homolog of p97, reveal that Cdc48 is also required for disassembly of mitotic spindles after execution of the mitotic exit pathway. Furthermore, Cdc48/p97-Ufd1-Npl4 directly binds to spindle assembly factors and regulates their interaction with microtubules at the end of mitosis. Therefore, Cdc48/p97-Ufd1-Npl4 is an essential chaperone that regulates transformation of the microtubule structure as cells reenter interphase.

Adenosine Triphosphatases↗
PubMed2003

Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP.

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

Amino Acid Sequence↗
PubMed2003

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal.

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

Animals↗
PubMed2003

A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo.

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.

3T3 Cells↗
PubMed2003

Ran in the spindle checkpoint: a new function for a versatile GTPase.

The small GTPase Ran has a well-established role in nucleocytoplasmic trafficking. In recent years, the repertoire of Ran has expanded to include regulation of spindle assembly, formation of the nuclear envelope and DNA replication. Now, new studies further extend the role of Ran to regulating the spindle checkpoint during mitosis.

Animals↗
PubMed2003

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly.

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.

Animals↗
PubMed2003

Characterization of a new gammaTuRC subunit with WD repeats.

The gamma-tubulin ring complex (gammaTuRC), consisting of multiple protein subunits, can nucleate microtubule assembly. Although many subunits of the gammaTuRC have been identified, a complete set remains to be defined in any organism. In addition, how the subunits interact with each other to assemble into gammaTuRC remains largely unknown. Here, we report the characterization of a novel gammaTuRC subunit, Drosophila gamma ring protein with WD repeats (Dgp71WD). With the exception of gamma-tubulin, Dgp71WD is the only gammaTuRC component identified to date that does not contain the grip motifs, which are signature sequences conserved in gammaTuRC components. By performing immunoprecipitations after pair-wise coexpression in Sf9 cells, we show that Dgp71WD directly interacts with the grip motif-containing gammaTuRC subunits, Dgrips84, 91, 128, and 163, suggesting that Dgp71WD may play a scaffolding role in gammaTuRC organization. We also show that Dgrips128 and 163, like Dgrips84 and 91, can interact directly with gamma-tubulin. Coexpression of any of these grip motif-containing proteins with gamma-tubulin promotes gamma-tubulin binding to guanine nucleotide. In contrast, in the same assay Dgp71WD interacts with gamma-tubulin but does not facilitate nucleotide binding.

Amino Acid Sequence↗