Ndc80复合体在外层动粒中的作用

动粒是参与有丝分裂过程中染色体分离的蛋白的附着支架。结构保守的Ndc80复合体位于动粒的外层,连接动粒和微管,与动粒-微管连接的稳定性有关。Aurora B/Ipl1激酶参与纠正动粒-微管的错误连接。Ndc80复合体对纺锤体组装检查点的功能非常重要。本文主要介绍了Ndc80复合体的研究进展。     

Studies of the Kinetochore Proteins of the Regenerating Liver and the Liver Cells of Rats at Different Stages of Development

The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.     

Influence of Monoclonal Antibodies on Microtubule Assembly

The influence on microtubule assembly in vitro of monoclonal antibodies against microtubule-associated proteins (MAPs) was studied. Light scattering was used for measuring net polymer formation and electron microscopy for determining the influence of antibodies on microtubule morphology. Control experiments showed that nonimmune mouse IgG had no effect on either the assembly or appearance of microtubules. The same was true for monoclonal antibodies against MAP1. At low levels, antibodies against MAP2 caused the aggregation of microtubules into bundles, an effect that did not occur with antibodies against any other MAP type studied. At increasing concentrations, anti-MAP2 progressively inhibited tubulin polymerization, producing irregular, shortened filaments. Anti-MAP5 produced a striking fragmentation of microtubules into very short pieces that were otherwise morphologically identical to control microtubules. The different effects of these antibodies show the potential of monoclonal antibodies for investigating MAP function and form an important adjunct to cellular microinjection experiments.     

TACC3 Regulates Microtubule Plus-End Dynamics and Cargo Transport in Interphase Cells

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Function and Assembly of DNA Looping,Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.     

Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.     

成熟促进因子对克隆重构胚核重编程的调控

成熟促进因子(maturation promoting factor,MPF)由催化亚单位P34cdc2和调节亚单位cyclin组成,对细胞周期的调控起着重要作用。目前,在核移植研究中发现：供体核在MPF的作用下发生核膜破裂(nuclear envelop breakdown．NEBD)和早熟染色体凝集(premature chromosome condensation,PCC),促进了核、质蛋白质因子的交换,有利于核重编程的进行。PCC还会对供体核的倍性及形态产生影响。     

Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes

We have previously shown that bovine oocytes parthenogenetically activated after 40 hours (hr) of in vitro maturation proceed through the cell cycle faster than those after 20 hr of maturation. In the present study, we used this model of different speed of nuclear progression to investigate the correlation of two hallmarks of nuclear events, exit of metaphase arrest and pronuclear formation, with dynamics of MPF and MAPK. Bovine oocytes were matured in vitro for 20 hr (young) or 40 hr (aged) and activated in 7% ethanol followed by incubation in cycloheximide for 0, 0.5, 1, 3, 5, or 7 hr. Activity of MPF and MAPK was lower in aged than young oocytes. The responses to oocyte activation by both the two kinases and nuclear progression were faster in aged than in young oocytes. The activity of MPF declined to undetectable levels (P < 0.05) as early as 0.5 hr after activation in aged oocytes, while this did not happen in young oocytes until 3 hr after activation. The inactivation of MAPK occurred approximately 2 hr earlier in aged oocytes (5 hr post-activation) than in young oocytes (7 hr post-activation). Furthermore, the decline in MPF activity preceded that of MAPK in both young and aged oocytes by about 2 hr. The decrease in activity of MPF and MAPK corresponded with the exit from meiosis and pronuclei formation regardless of the speed of nuclear progression. Despite dramatic changes in activity of MPF and MAPK, the levels of Cdc2 and Erk2 proteins were unchanged (P > 0.05) during the first 7 hr of activation. These observations suggest that inactivation of MPF and MAPK are pre-requisite for the release from metaphase arrest and formation of pronuclei in bovine oocytes.     

Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 microsec). After the artificial activation, 40-50 oocytes were cultured in a 50 microl drop of NCSU23 medium supplemented with 0.4% BSA at 39 degrees C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 microsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 microsec) (P < 0.05). Single electrical stimulus (180 V/mm, 15 microsec) resulted in higher preimplantation development of porcine oocytes as compared to other chemical stimulators (P < 0.01). Western blot analyses showed the decrease of MPF and MAP kinase in the electrically-activated oocytes. After single electrical stimulus, the amounts of both cdc2 and ERK in porcine oocytes were remarkably reduced by 4 hr and then further decreased by 8 hr. However, the chemically-stimulated oocytes did not show any significant change at the levels of MPF and MAP kinase. Our results indicate that the optimal single electrical pulse is effective on the inactivation of MPF and MAP kinase, eventually leading to the parthenogenetic development of porcine oocytes.     

Microtubule Attachment and Centromeric Tension Shape the Protein Architecture of the Human Kinetochore

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Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.     

Epigenetic specification of centromeres by CENP-A

Centromeres are the chromosomal loci that direct the formation of the kinetochores. These macromolecular assemblies mediate the interaction between chromosomes and spindle microtubules and thereby power chromosome movement during cell division. They are also the sites of extensive regulation of the chromosome segregation process. Except in the case of budding yeast, centromere identity does not rely on DNA sequence but on the presence of a special nucleosome that contains a histone H3 variant known as CenH3 or CENP-A (Centromere Protein A). It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Upon DNA replication the mark is diluted two-fold and must be replenished to maintain centromere identity. What distinguishes CENP-A nucleosomes from those containing histone H3, how CENP-A nucleosomes are incorporated specifically into centromeric chromatin, and how this incorporation is coordinated with other cell cycle events are key issues that have been the focus of intensive research over the last decade. Here we review some of the highlights of this research.     

Promotion of Microtubule Assembly by Neurofilament-Associated Microtubule-Associated Proteins

Abstract: Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.     

Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.     

Phosphorylation at Threonine 288 by Cell Cycle Checkpoint Kinase 2 (CHK2) Controls Human Monopolar Spindle 1 (Mps1) Kinetochore Localization

Human Mps1 (hMps1) is a mitotic checkpoint kinase responsible for sensing the unattached and tensionless kinetochore. Despite its importance in safeguarding proper chromosome segregation, how hMps1 is recruited to the kinetochore remains incompletely understood. Here, we demonstrate that phosphorylation at Thr-288 by the cell cycle checkpoint kinase CHK2 is involved in this process. We discovered that the phosphorylation-deficient T288A mutant has an impaired ability to localize to the kinetochore and cannot reestablish the mitotic checkpoint in hMps1-depleted cells. In support, we found that nocodazole induced hMps1 phosphorylation at the previously identified CHK2 site Thr-288 and that this could be detected at the kinetochore in a CHK2-dependent manner. Mechanistically, phosphorylation at Thr-288 promoted the interaction with the KMN (KNL1-Mis12-Ndc80 network) protein HEC1. Forced kinetochore localization corrected the defects associated with the T288A mutant. Our results provide evidence of a newly identified hMps1 phosphorylation site that is involved in the mitotic checkpoint and that CHK2 contributes to chromosomal stability through hMps1.     

Visualization and Molecular Analysis of Actin Assembly in Living Cells

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.