DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation

We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin- related proteins. Immunoblots using an antibody raised against a non- conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone.     

Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA- damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.     

The protease activity of yeast separase (esp1) is required for anaphase spindle elongation independently of its role in cleavage of cohesin

Separase is a caspase-family protease required for the metaphase-anaphase transition in eukaryotes. In budding yeast, the separase ortholog, Esp1, has been shown to cleave a subunit of cohesin, Mcd1 (Scc1), thereby releasing sister chromatids from cohesion and allowing anaphase. However, whether Esp1 has other substrates required for anaphase has been controversial. Whereas it has been reported that cleavage of Mcd1 is sufficient to trigger anaphase in the absence of Esp1 activation, another study using a temperature-sensitive esp1 mutant concluded that depletion of Mcd1 was not sufficient for anaphase in the absence of Esp1 function. Here we revisit the issue and demonstrate that neither depletion of Mcd1 nor ectopic cleavage of Mcd1 by Tev1 protease is sufficient to support anaphase in an esp1 temperature-sensitive mutant. Furthermore, we demonstrate that the catalytic activity of the Esp1 protease is required for this Mcd1-independent anaphase function. These data suggest that another protein, possibly a spindle-associated protein, is cleaved by Esp1 to allow anaphase. Such a function is consistent with the previous observation that Esp1 localizes to the mitotic spindle during anaphase.     

The budding yeast GSK-3 homologue Mck1 is an essential component of the spindle position checkpoint

The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1''s function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

A Highlights from MBoC Selection: The kinesin-14 Klp2 is negatively regulated by the SIN for proper spindle elongation and telophase nuclear positioning

In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end–directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized.     

A novel role for the mitotic spindle during DNA segregation in yeast: promoting 2 microm plasmid-cohesin association

The 2 microm circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal "selfish" DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2 microm circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.     

The myokine decorin is regulated by contraction and involved in muscle hypertrophy

The health-promoting effects of regular exercise are well known, and myokines may mediate some of these effects. The small leucine-rich proteoglycan decorin has been described as a myokine for some time. However, its regulation and impact on skeletal muscle has not been investigated in detail. In this study, we report decorin to be differentially expressed and released in response to muscle contraction using different approaches. Decorin is released from contracting human myotubes, and circulating decorin levels are increased in response to acute resistance exercise in humans. Moreover, decorin expression in skeletal muscle is increased in humans and mice after chronic training. Because decorin directly binds myostatin, a potent inhibitor of muscle growth, we investigated a potential function of decorin in the regulation of skeletal muscle growth. In vivo overexpression of decorin in murine skeletal muscle promoted expression of the pro-myogenic factor Mighty, which is negatively regulated by myostatin. We also found Myod1 and follistatin to be increased in response to decorin overexpression. Moreover, muscle-specific ubiquitin ligases atrogin1 and MuRF1, which are involved in atrophic pathways, were reduced by decorin overexpression. In summary, our findings suggest that decorin secreted from myotubes in response to exercise is involved in the regulation of muscle hypertrophy and hence could play a role in exercise-related restructuring processes of skeletal muscle.     

不同频率慢性电刺激膈神经对兔膈肌骨骼肌型二氢吡啶受体α1亚单位、ryanodine受体mRNA和蛋白表达的影响

测定不同频率慢性电刺激(chronic electrical stimulation,CES)膈神经5周后对兔膈肌钙释放单位中骨骼肌型二氢吡啶受体(DHPR)α1亚单位和ryanodine受体(RyRs)的mRNA和蛋白表达水平的影响,探讨CES后兔膈肌钙释放单位结构组成的变化和可能的临床应用价值.封闭群日本大耳白兔30只,随机分为正常对照组、10、20、50和100Hz,每组6只;以10和20 Hz为慢性低频电刺激组,50和100Hz为慢性高频电刺激组.CES参数为波宽0.2 ms 3～6个波/次,45次/min,电压10～20 V.刺激时间2×2 h/日,每周刺激6 d,连续刺激5周.分别采用RT-PCR和免疫组织化学法测定兔膈肌骨骼肌型DHPRα1-亚单位和RyR1、RyR2和RyR3的mRNA和蛋白表达.结果显示与对照组比较,慢性低频电刺激10和20 Hz组骨骼肌型DHPRα1、RyR的mRNA和蛋白表达明显降低(P＜0.01),有低度的RyR,mRNA的表达出现;慢性高频电刺激50和100Hz组骨骼肌型DHPRα1、RyR1的mRNA和蛋白表达明显升高(P＜0.01),未检测到RyR2mRNA的阳性表达.本实验提示慢性低频电刺激膈神经5周后,膈肌质膜上DHPR与RyRs之间的信号转导方式已从变构耦联为主转变为以Ca2+诱导Ca2+释放耦联为主.     

A cell cycle role for a plant sucrose nonfermenting-1-related protein kinase (SnRK1) is indicated by expression in yeast

Plant sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRK1s) have been shown to restore carbon catabolite derepression of gene expression in the yeast Saccharomyces cerevisiae when expressed in snf1 mutants. SNF1 has been implicated in the mediation of cell cycle control in response to nutrient levels and, in the present study, we show that expression of the rye (Secale cereale) SnRK1, RKIN1, in a yeast snf1 mutant has a dramatic effect on the size of cells growing on a minimal medium where SNF1 function is essential. The mean volume of the yeast cells which were expressing RKIN1 was two-thirds that of the whi1 mutant, the smallest viable cells known in S. cerevisiae, and the cells died after 3 days unless rescued onto complex medium. This is the first experimental evidence of a role for SnRK1s in plant cell cycle control.     

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

Interaction of human MUC1 and beta-catenin is regulated by Lck and ZAP-70 in activated Jurkat T cells

The MUC1 transmembrane glycoprotein is aberrantly expressed by diverse hematologic malignancies, including those of the T cell lineage. The MUC1 cytoplasmic domain (CD) interacts with beta-catenin; however, the role of MUC1 in T cells is not known. In the present work, MUC1 was studied as a potential downstream effector of the Lck and ZAP-70 tyrosine kinases that are essential for T cell activation. The results demonstrate that anti-CD3-induced or PMA+ionomycin-induced activation of Jurkat T cells is associated with increased binding of MUC1 and Lck. Lck phosphorylates MUC1-CD on Y-46 and, in turn, stimulates the binding of MUC1 to beta-catenin. The results further demonstrate that MUC1 interacts with ZAP-70. In contrast to Lck, ZAP-70 phosphorylates MUC1-CD predominantly on Y-20. However, like Lck, ZAP-70-mediated phosphorylation of MUC1 Y-20 stimulates binding of MUC1 and beta-catenin. These findings indicate that MUC1 functions as a substrate for Lck and ZAP-70 in activated Jurkat T cells and that MUC1 integrates T cell receptor signaling with the beta-catenin pathway.