酵母纺锤体极体复制缺陷与染色体不稳定性的发生

纺锤体极体作为酵母细胞的微管组织中心,在功能上等同于高等真核细胞的中心体,它在细胞周期中的准确复制是两极纺锤体组装和染色体正确分离的前提。纺锤体极体复制缺陷会导致异倍体和多倍体的形成,造成染色体不稳定性的发生。以酿酒酵母细胞为模型,研究纺锤体极体复制过程相关蛋白质的突变,有助于揭示酵母细胞中染色体不稳定性发生的分子机制,并为动物细胞中心体复制的研究提供良好的借鉴。     

酵母Bir1p同源物Survivin对毕赤酵母的分子重组与生长影响

Bir1p是酵母凋亡途径中抑制细胞凋亡的重要蛋白,Survivin是Bir1p的人源同源物,Survivin第34位氨基酸T向A的突变能使其功能逆转.将Survivin及其突变体Survivin(T34A)的基因分别构建到组成型穿梭表达质粒pGAPZA中,整合入毕赤酵母的基因组,分析对酵母细胞凋亡的影响.酵母生长曲线、MTT及流式细胞仪测定数据显示,Survivin和其突变体基因在酵母中的表达分别抑制和促进了酵母凋亡.多样的工艺对工业用酵母的凋亡提出了不同的要求,本研究为工程酵母的理性改造提供了理论依据.     

DNA损伤反应性蛋白参与中心体调控

中心体是动物细胞有丝分裂期微管组织中心,对于细胞有丝分裂期形成纺锤体、正常分裂及染色体精确分离至关重要. 中心体失调控常造成遗传物质错误分配,最终诱发肿瘤形成.因此,对中心体结构及数量的精密调控将对细胞命运起着决定 作用.目前发现,中心体至少包含100多种调节蛋白,这些蛋白在细胞内的功能各异.最近很多研究显示,多种DNA损伤修复及 应答通路的激酶或磷酸酶定位于中心体,并且参与中心体调控.本文将对中心体结构、中心体复制、中心体分离、中心体扩 增、DNA损伤与中心体异常及DNA损伤反应性蛋白在中心体调控中的功能作一综述.     

利用人源化酵母系统模拟tomosyn在囊泡运输过程中的功能

突触囊泡融合是由syntaxin-1A, synaptobrevin-2和突触小体相关蛋白25(SNAP-25)三个突触N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)蛋白共同介导完成的.这些突触SNARE蛋白之间的相互作用受到包括tomosyn在内的多种辅助蛋白的调控. tomosyn是一个syntaxin结合蛋白,并且它是一个突触囊泡融合的负调控因子.然而, tomosyn的具体作用方式并不完全清楚.本研究在酵母细胞中重构了tomosyn的抑制作用模型. SNARE蛋白是一种保守的蛋白,在酵母中,突触SNARE蛋白的同源蛋白参与了分泌囊泡和细胞膜的融合. Sso1是酵母中syntaxin-1A的同源蛋白.在之前的实验中,我们构建了一个SNARE嵌合体,命名为Sso1/187k-STX1A~(D133V),其中将Sso1的一部分替换为syntaxin-1A的相应部分.这个SNARE嵌合体可以取代Sso1在酵母细胞中的功能.过表达tomosyn对野生型酵母细胞的生长没有影响.然而, tomosyn的过表达干扰了含有Sso1/187K-STX1A~(D133V)的细胞的生长.酵母双杂交试验结果表明,在酵母细胞中, tomosyn与syntaxin相互作用,而不是与Sso1相互作用.因此,我们可以使用含有Sso1/187K-STX1A~(D133V)的酵母细胞作为分析tomosyn功能的工具.     

多功能蛋白Geminin的研究进展

Geminin是一种定位于核内的多功能小分子蛋白,具有相对复杂的结构模式,在细胞增殖、胚胎发育及肿瘤发生等多方面均发挥重要作用.它通过调节细胞周期时相中的重要事件作用于细胞增殖:经多种途径参与DNA复制的调节;抑制中心体重复复制;推进G2/M期和维持正常胞质分裂等.在不同发育阶段,Geminin可作为抑制因子或是诱导因子参与胚胎发育的调节,特别是在神经形成方面.通过与同源异型盒基因或蛋白Six3及Hox等的相互作用,Geminin分别在眼睛发育及胚胎发育过程中起调节作用,并且表现出在细胞增殖与分化中协调因子的功能.近年来Geminin在肿瘤中的作用已成为研究的重点,它可作为评价肿瘤发生进程和预后的标志分子,并可望是一个新的肿瘤治疗的作用靶点.对Geminin活性的调节主要表现在转录水平和转录后水平,转录后水平的调节可能占主要地位.     

小麦RAN1在酵母系统中影响微管的完整性和核质运输过程

真核生物的小G蛋白Ran在进化过程中比较保守,它可直接参与细胞周期调控过程,它的缺失突变可以影响很多细胞生理进程.我们已经从小麦(Triticum aestivum L.cv.Jingdong No.1)cDNA文库中克隆到一个新的RanGTPase的同源基因TaRAN1.在此基础上利用裂殖酵母模式系统研究了该基因的功能.研究结果表明,TaRAN1基因超表达可产生缺陷的纺锤体微管,这可能是导致我们以前观察到的异常染色体分离现象的原因.反义TaRAN1基因表达的酵母细胞,微管系统受到破坏.我们推测TaRAN1蛋白在细胞有丝分裂的纺锤体组装和维持微管系统的完整与稳定过程中起着重要作用.透射电镜观察实验结果显示,超表达TaRAN1的酵母细胞具有异常的核膜结构,反义表达TaRAN1的酵母细胞有异常的液泡结构和紊乱的膜结构,由此推测,TaRAN1在整个核质运输事件中可能是必须的.     

中心体蛋白centrin研究进展

Centrin是普遍存在于中心体上的蛋白成分 ,具有钙离子结合能力。Centrin家族包含多种同源蛋白 ,这些蛋白在结构上高度保守。研究表明centrin可能与细胞的分化 ,纤毛的生成定向和切断 ,精子尾部的生成 ,中心体复制及其结构维持有密切关系 ,同时也和细胞的癌变、生长及细胞周期调控有关 ,还有许多未知功能有待研究     

与丙型肝炎病毒复制中间体3′末端结合的宿主蛋白的研究

宿主细胞蛋白在HCV复制过程中起着重要的作用.应用蛋白质-核酸紫外交联法检测多个细胞株,目的是明确是否存在可与HCV复制中间体3′末端特异性结合的细胞蛋白质.结果显示,在多个细胞株中存在一个分子质量约为45 ku的蛋白质（命名为p45）,可与HCV复制中间体3′末端131～278 nt结合,这种结合可被过量的自身未标记RNA竞争抑制,而非同源蛋白和非同源RNA均不能竞争抑制这种结合.根据计算机RNA二级结构分析推测,p45可能特异性结合于HCV复制中间体3′端的一茎环结构内.这一结果提示,p45在HCV子代RNA复制中可能起着重要作用.     

小麦RAN1在酵母系统中影响微管的完整性和核质运输过程

真核生物的小G蛋白 Ran在进化过程中比较保守,它可直接参与细胞周期调控过程,它的缺失突变可以影响很多细胞生理进程。我们已经从小麦(Triticum aestivum L. cv. Jingdong No. 1) cDNA文库中克隆到一个新的RanGTPase的同源基因TaRAN1。在此基础上利用裂殖酵母模式系统研究了该基因的功能。研究结果表明,TaRAN1基因超表达可产生缺陷的纺锤体微管,这可能是导致我们以前观察到的异常染色体分离现象的原因。反义TaRAN1基因表达的酵母细胞,微管系统受到破坏。我们推测TaRAN1蛋白在细胞有丝分裂的纺锤体组装和维持微管系统的完整与稳定过程中起着重要作用。透射电镜观察实验结果显示, 超表达TaRAN1的酵母细胞具有异常的核膜结构,反义表达TaRAN1的酵母细胞有异常的液泡结构和紊乱的膜结构,由此推测, TaRAN1在整个核质运输事件中可能是必须的。     

p62蛋白功能及相关信号通路研究

p62是一种多功能蛋白,其蛋白分子包含多个结构域,通过与不同蛋白质结合形成细胞中重要的信号中心,从而调控多种信号通路,影响细胞的生长、衰老,甚至死亡等生理过程。p62蛋白通过对mTORC1信号通路的影响在氨基酸信号通路中发挥着关键的调控作用。p62蛋白是自噬体与底物之间的适配蛋白,在细胞自噬过程中起到分子调节器的作用。p62蛋白具有质核穿梭功能,在DNA损伤修复和氧化应激反应中具有重要作用,其异常积累会引起细胞的恶性转变,导致肿瘤的发生。现综述p62在调节多种信号通路,如自噬、氨基酸感知、凋亡及肿瘤发生等过程中的作用。     

Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p-centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous alpha helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p-centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.     

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.     

Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.     

Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1.     

Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.     

Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

A role for centrin 3 in centrosome reproduction

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Transient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s). Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.     

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.     

Centrosome Duplication: Is Asymmetry the Clue?

The structure of the yeast Sfi1-centrin complex, and its asymmetric position within the yeast centrosome, suggest a model for the initiation of centrosome duplication and provides a target for licensing this event.     

MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication

It is crucial to the eucaryotic cell cycle that the centrosome undergo precise duplication to generate the two poles of the mitotic spindle. In the budding yeast Saccharomyces cerevisiae, centrosomal functions are provided by the spindle pole body (SPB), which is duplicated at the time of bud emergence in G1 of the cell cycle. Genetic control of this process has previously been revealed by the characterization of mutants in CDC31 and KAR1, which prevent SPB duplication and lead to formation of a monopolar spindle. Newly isolated mutations described here (mps1 and mps2, for monopolar spindle) similarly cause monopolar mitosis but their underlying effects on SPB duplication are unique. The MPS1 gene is found by electron microscopy to be essential for proper formation of the site at which the new SPB normally arises adjacent to the existing one. By contrast, a mutation in MPS2 permits duplication to proceed, but the newly formed SPB is structurally defective and unable to serve as a functional spindle pole. Distinct temporal requirements for the CDC31, MPS1, and MPS2 gene functions during the SPB duplication cycle further demonstrate the individual roles of these genes in the morphogenetic pathway.