植物胞质分裂发生机制

胞质分裂(cytohnesis)是指在同一细胞中在新形成的两个子核之间形成新的间隔,将母细胞一分为二的过程。胞质分裂存在于任何一种生命形式中,从单细胞的细菌到多细胞的真核生物都能进行胞质分裂。近些年由于细胞学方法的改进和研究材料增多等因素,使得对植物胞质分裂发生机制的研究取得了很大的进展。现对植物中不同类型的胞质分裂在细胞学、分子生物学方面的研究进展作一综述。     

朱顶红生殖细胞胞质分裂的两种方式

大多数植物以形成细胞板方式完成胞质分裂过程,也有些植物以类似于动物和单细胞植物在赤道区形成收缩沟的方式而分成两部分。本工作应用电镜对朱顶红体外萌发9-18小时花粉管中的生殖细胞胞质分裂进行了研究。结果表明：70％的细胞表现的是第一种方式,30％却是第二种方式。即：朱硕红生殖细胞胞质分裂同时存在两种方式。前者最初以细胞板亚单位的形式出现于有丝分裂晚后期,它们聚集于成膜体的中央区域并于分裂末期融合成一个大的连续的单位(Fig．1-3)。大量新的微管形成于两组染色体之间(Fig．1)。分裂末期,细胞板形成并具胞质通道(Fig.2)。成膜体微管规则排列并穿过胞质通道向新形成的末期核伸展(Fig.2＆3)。这些微管与构成细胞板的质膜紧密联系(Fig．3)。后者则在有丝分裂后期开始(Fig．4),当两群染色体彼此分离时,生殖细胞质膜在中央区由两侧向内凹陷形成收缩沟。有时生殖细胞几乎被收缩沟分成两个部分(Fig．6)。发生缢缩的细胞中细胞器与具细胞板的无差异,但微管稀少并且排列紊乱(Fig．4＆5),染色体的状态使得难以准确区分细胞分裂时期。而且核膜的形成似乎始于有丝分裂后期、出现于染色体边缘(Fig.7)。有时尚有落后染色体出现(Fig.8)。据此认为：收缩沟的发生与核膜的重建、染色体的异常行为及微管无序有关。朱顶红生殖细胞同时存在两种方式的胞质分裂现象相当特殊,可能存在着两种胞质分裂机制。由于游离的生殖细胞在某种程度上类似于动物细胞,因而以缢缩方式完成胞质分裂是可能的。另一方面,生殖细胞对花粉管生长所处的环境极为敏感,体外培养造成生殖细胞不规剧分裂的可能性也应考虑。因此研究在柱头上萌发花粉管中的生殖细胞的胞质分裂是有意义的,此研究结果将有助于更好地理解生殖细胞胞质分裂的机制。     

裂殖酵母Cnb1参与胞质分裂过程

丝/苏氨酸特异性钙调磷酸酶(Calcineurin, CN)是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子。裂殖酵母中的CN是由催化亚基Ppb1和调节亚基Cnb1组成的异源二聚体。文章报道了裂殖酵母中cnb1+的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞。胞质分裂过程中, Cnb1与Ppb1组成CN复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩。cnb1Δ菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象。上述结果说明Cnb1可能参与隔膜的成熟过程。此外, 还检测了cnb1D菌株中胞裂蛋白的信号。胞裂蛋白包括Spn1、Spn2、Spn3和Spn4, 它们是引导隔膜降解的重要分子。结果显示, 在cnb1D菌株中, 80%左右的细胞在隔膜处缺失Spn2和Spn3的信号, 20%左右的细胞缺失Spn1和Spn4的信号。由于胞裂蛋白的蛋白表达量在cnb1D中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示Cnb1可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性。以上结果提示, Cnb1可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程。     

MAPK级联途径调控植物细胞胞质分裂

胞质分裂(cytokinesis)是细胞分裂的最后关键一步,产生2个含有完整的遗传物质和胞质细胞器的子细胞．植物胞质分裂包括细胞板的形成,这一过程是在成膜体的牵引下由一些植物特有的步骤完成的．促分裂原活化蛋白激酶(MAPK)级联途径在真核生物中是高度保守的,由MAPKs,MAPKKs,MAPKKKs组成,通过MAPKKK→ MAPKK → MAPK的逐级磷酸化传递细胞信号．近来的研究表明, NACK-MAPKKK→MAPKK→MAPK→MAP65构成的信号途径调控植物细胞的胞质分裂．本文就这一信号途径,总结了植物胞质分裂机制的研究进展,并对其中的问题进行了讨论与展望．     

胞质分裂阻滞细胞的微核检测及方法学初步研究

培养人体外周血淋巴细胞的微核检测,是近年来常用的体外短期检测方法之一,可用来评价被检理化因子对人体细胞的遗传毒性,及监测环境、职业接触等有害因子对人类的潜在致癌作用。为了进一步提高该方法的敏感性,根据微核起源于染色体断片与落后染色体的观     

钙调素（CaM）在中体上的分布及参与胞质分裂的调控

钙调素(CaM)是细胞内Ca^2 的主要受体,在细胞增殖、分化、凋亡、迁移等过程中都发挥着重要的调控作用。采用GFP标记技术,我们观察了GFP—CaM在胞质分裂期HeLa细胞中的动态分布,发现在胞质分裂后期,GFP—CaM与中体紧密相连。抑制CaM的活性会阻止中体的解聚。进一步观察发现,CaM与γ-微管蛋白共分布在中体两侧,抑制CaM活性也会引起中体γ-微管蛋白解离的延迟。本实验结果说明分布在中体上的CaM很可能通过影响中体微管的稳定,参与调控胞质分裂的完成。     

Cdc42和球形肌动蛋白在卵母细胞胞质分裂中的定位分析

研究活性Cdc42与球形肌动蛋白(G-actin)在爪蟾卵母细胞胞质分裂中的定位关系。分别用GFP-wGBDmRNA与罗丹明-594-微管蛋白、Alexa-488-球形肌动蛋白与罗丹明-594-微管蛋白、GFP-wGBDmRNA与Alexa-594-球形肌动蛋白共同显微注射爪蟾卵母细胞。利用共聚焦显微镜,时间延迟摄影方法,分别观察活体卵母细胞中活性Cdc42、球形肌动蛋白在胞质分裂过程中的定位,以及活性Cdc42与球形肌动蛋白在胞质分裂中的定位关系。在卵母细胞胞质分裂中,活性Cdc42与球形肌动蛋白存在空间上共定位现象,并且在时相上具有一致性。结果提示活性Cdc42和球形肌动蛋白在卵母细胞胞质分裂过程中密切相关。     

esiRNA分析人源驱动蛋白MKLP1在胞质分裂中的功能

为探讨人源驱动蛋白MKLP1在有丝分裂和胞质分裂中的作用,以E.coliRNaseⅢ制备MKLP1的3′UTResiRNA转染HeLa细胞,通过定量RTPCR、Western印迹检测MKLP1esiRNA对MKLP1基因的沉默效率.再利用FACS分析、免疫荧光染色和活细胞成像分析检测MKLP1表达缺失后在有丝分裂和胞质分裂不同时期的细胞形态学、细胞分裂指数、细胞百分数,动态观察有丝分裂和胞质分裂期间的表型改变,以系统分析MKLP1的功能.最后通过挽救实验验证MKLP1esiRNA的作用特异性.实验显示MKLP1esiRNA转染HeLa细胞能够有效地特异性消除MKLP1的表达,并被异位表达的MKLP1所挽救.MKLP1蛋白在有丝分裂后期和末期前期位于纺锤体中间带,在末期后期和胞质分裂的最后阶段集中于中间体的中心处.MKLP1表达缺失使中间体正确形成和胞质分裂的完成受到严重抑制,造成大量双多核细胞堆积.结果表明,MKLP1在胞质分裂中间体形成和有丝分裂末期前期向后期过渡过程中起关键作用,是纺锤体中间体中间带相关蛋白,为胞质分裂所必需.     

钙调素(CaM)在中体上的分布及参与胞质分裂的调控

钙调素(CaM)是细胞内Ca~(2+)的主要受体,在细胞增殖、分化、凋亡、迁移等过程中都发挥着重要的调控作用。采用GFP标记技术,我们观察了GFP-CaM在胞质分裂期HeLa细胞中的动态分布,发现在胞质分裂后期,GFP-CaM与中体紧密相连。抑制CaM的活性会阻止中体的解聚。进一步观察发现,CaM与γ-微管蛋白共分布在中体两侧,抑制CaM活性也会引起中体γ-微管蛋白解离的延迟。本实验结果说明分布在中体上的CaM很可能通过影响中体微管的稳定,参与调控胞质分裂的完成。     

叶绿体分裂蛋白PDV1胞质侧结构域可溶性表达条件的研究及纯化

目的:探索叶绿体分裂蛋白PLASTID DIVISION1(PDV1)胞质侧结构域的高效可溶性表达条件,并得到高纯度目的蛋白。方法:通过改变表达载体种类、基因片段大小、诱导剂浓度、诱导温度的方法,以及运用分子伴侣的协助,实现目的蛋白高效可溶性表达。通过镍柱亲和层析和分子筛层析纯化目的蛋白。结果:(1)带His标签的目的蛋白大部分以包涵体形式存在于沉淀中;(2)截掉疏水区域并与增溶标签GST或NusA融合表达,再通过改变诱导表达条件,可以实现PDV1胞质侧结构域的可溶性表达;(3)比较目的蛋白可溶性表达量,选择高效可溶性表达体系,并在该条件下纯化得到高纯度目的蛋白。结论:PDV1胞质侧结构域的高效可溶性表达及纯化,为进一步研究该蛋白的结构及其在叶绿体分裂过程中的作用奠定了一定基础。     

Characterization of centrosomal proteins Cep55 and pericentrin in intercellular bridges of mouse testes

Centrosomal protein 55 (Cep55), located in the centrosome in interphase cells and recruited to the midbody during cytokinesis, is essential for completion of cell abscission. Northern blot previously showed that a high level of Cep55 is predominantly expressed in the testis. In the present study, we examined the spatial and temporal expression patterns of Cep55 during mouse testis maturation. We found that Cep55, together with pericentrin, another centrosomal protein, were localized to the intercellular bridges (IBs) interconnecting spermatogenic cells in a syncytium. The IBs were elaborated as a double ring structure formed by an inner ring decorated by Cep55 or pericentrin and an outer ring of mitotic kinesin‐like protein 1 (MKLP1) in the male germ cell in early postnatal stages and adulthood. In addition, Cep55 and pericentrin were also localized to the acrosome region and flagellum neck and middle piece in elongated spermatids, respectively. These results suggest that Cep55 and pericentrin are required for the stable bridge between germ cells during spermatogenesis and spermiogenesis. J. Cell. Biochem. 109: 1274–1285, 2010. © 2010 Wiley‐Liss, Inc.     

SPG20 Protein Spartin Is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1–7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin–Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.     

The Centriolar Protein Bld10/Cep135 Is Required to Establish Centrosome Asymmetry in Drosophila Neuroblasts

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Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site.     

Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation

Microtubule‐nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The γ‐tubulin ring complexes (γTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled‐coil proteins, pericentrin/kendrin and CG‐NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule‐organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna‐interacting protein. Interestingly, Cep72 is essential for the localization of CG‐NAP and Kizuna. Cep72 is also involved in γTuRC recruitment to the centrosome and CG‐NAP confers the microtubule‐nucleation activity on the γTuRCs. During mitosis, Cep72‐mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule‐organizing activity and structural integrity of the centrosome.     

Centrosome/Spindle Pole–associated Protein Regulates Cytokinesis via Promoting the Recruitment of MyoGEF to the Central Spindle

Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi interferes with MyoGEF localization at the central spindle. Finally, MyoGEF interacts with ECT2, and RNAi-mediated depletion of MyoGEF leads to mislocalization of ECT2 and RhoA during cytokinesis. Therefore, we propose that CSPP interacts with and recruits MyoGEF to the central spindle, where MyoGEF contributes to the spatiotemporal regulation of cytokinesis.     

人源CT55蛋白原核表达及单克隆抗体的制备

为制备Cancer testis 55(CT55)单克隆抗体,需构建带有人源CT55片段的原核表达质粒,把该质粒转化Rosetta感受态进行原核表达并得到目的蛋白,蛋白质被纯化后免疫6周雌性BALB/c小鼠。按传统的单克隆抗体的制备方法,取小鼠脾细胞与骨髓瘤细胞(sp2/0)进行融合,经ELISA方法筛选及两次连续亚克隆,共获得多株能稳定分泌抗CT55蛋白单克隆抗体的杂交瘤细胞,如3D8B7B12、4C8E1C9、3D8C10G9等。ELISA及Western blot(WB)分析结果表明,筛选的细胞株均能产生单克隆抗体,且该抗体均分别能与原核表达及真核表达的CT55蛋白发生特异性结合。单克隆抗体可用于免疫荧光试验,且与P53发生互作的荧光主要位于细胞核边缘。结果表明,成功制备了针对人源CT55蛋白的单克隆抗体。CT55蛋白单克隆抗体的制备为今后肝癌、胃癌、结肠癌等癌症的快速的病原学诊断以及CT55蛋白的结构和功能研究奠定了物质基础。     

RABL6A,a Novel RAB-Like Protein,Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.