p21活化蛋白激酶2在卵母细胞成熟中的作用

研究p21活化蛋白激酶2（p21-activated kinase 2,PAK2）在爪蟾卵母细胞成熟中的作用。利用特异性抑制PAK2活性的PAK2-N端（PAK2-N terminal,PAK2-NT）片段显微注射爪蟾卵母细胞。荧光显微镜下比较PAK2-NT mRNA注射组和未注射对照组卵母细胞胚泡破裂发生。共聚焦显微镜下,时间延迟摄影法观察两组卵母细胞胞质分裂过程中肌动蛋白和纺锤体的变化。与未注射PAK2-N端mRNA的对照组卵母细胞相比,注射组卵母细胞胚泡破裂发生无异常,但未见胞质分裂发生和极体形成。结果提示PAK2可能参与爪蟾卵母细胞胞质分裂过程。     

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。     

p21活化激酶的生物学活性及其与肿瘤的关系

p21活化激酶(p21-activatedkinase,PAK),为一类进化上保守的丝氨酸/苏氨酸蛋白激酶。PAK在许多组织中广泛表达,作为小G蛋白Rho家族Cdc42和Rac1的下游靶蛋白,可以被生长因子及其他胞外信号通过GTP酶依赖的信号通路或非GTP酶依赖的信号通路活化,发挥多种生物学效应。PAK作为一种重要的生物学调节因子,在哺乳动物一系列细胞功能中具有重要作用,如:细胞运动、细胞生存、细胞周期、血管生成、基因转录调节及癌细胞的侵袭转移。通过对PAK家族成员信号转导机制的研究,为癌症治疗提供分子靶标。     

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。     

监测活细胞中诱导激活的Rac、Cdc42时空图像的FRET单分子探针的构建

以PCR方法从人脑cDNA基因文库扩增Rac1、Cdc42 cDNA全序列及其效应蛋白基因Pak1、N-WASP的GTP酶联结区域(GBD)序列,从dsRed1-N1质粒扩增红色荧光蛋白dsRed1cDNA全序列.将cDNA序列依次定向克隆至pECFP-N1质粒载体,获得基于FRET原理,包含dsRed1,Pak1或N-WASP的GBD,Rac1或Cdc42,ECFP编码序列的单分子探针.在dsRed1的C末端加入一段CAAM法尼基化基序,构建包含EGFP,Pak1的GBD,Rac1或Cdc42,dsRed1-CAAM的质膜特异表达的单分子探针.采用这两种探针,可用于监测活细胞中诱导激活的Rac1、Cdc42信号转导通路的3D时空图像,检测待测蛋白分子的GEF或GAP活性.     

监测活细胞中诱导激活的Rac、Cdc42信号转导通路的FRET单分子探针

Rho家族鸟苷三磷酸酶,包括Rac1和Cdc42等．参与调节细胞形态、细胞迁移、转录激活和基因表达等一系列细胞过程。根据FRET（Fluorescent resonance energy transfer）技术原理,构建了包含红色荧光蛋白dsRed1与青色荧光蛋白ECFP的全长cDNA．效应分子Pak1或N—WASP的GTP酶联结区域．信号分子Rac1或Cdc42的全长cDNA的几种单分子探针。转染NIH3T3或Hela细胞,以胰岛素、缓激肽为诱导剂,分别激活Rac1、Cdc42信号转导通路。离体荧光光谱检测表明．在两种转染动物细胞中均产生了FRET现象。不同信号转导通路的FRET效率,在诱导激活5min后,均达到最高值,但增加幅度有显著差异。随着诱导时间的延长,FRET效率下降,但下降速率在不同信号转导通路间差异显著。Rac1、Cdc42激活试验证实．诱导激活的转染细胞中,Rac1、Cdc42均处于激活状态（GTP—bound）,其在不同诱导时间的相对激活程度与FRET效率表现相同。诱导激活的Rac1、Cdc42信号转导通路,分别导致了转染活细胞中片状伪足、线状伪足的产生。这表明,采用这些单分子探针,可直接监测激活的信号转导通路,在活细胞中的3D时空分布变化图像及其产生的细胞学效应。采用这些单分子探针．分析、判断了一些调节蛋白对Rac1、Cdc42的GEF或GAP特性。从而提供一种可大大简化现有的鉴定待测蛋白分子的方法。     

Rac1、Cdc42单分子探针在活细胞中表达差异的比较

利用构建的Rac1、Cdc42单分子探针,探索其在NIH3T3、Hela、COS7等活细胞中的最佳表达效果.结果表明在胞浆中表达的探针其荧光值在诱导5～10 min达到最大值,随着时间的推移,其荧光强度逐渐减弱;在细胞膜上表达的探针也是相似的.DNA/转染试剂为1:3时的表达效果最佳;每毫升转染培养基的质粒DNA含量为4或6 μg时,其转染表达可达到最佳;在不同种类的细胞中表达时其差异不明显;在细胞的不同部位表达差异不明显.     

Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1

Cell-cell contacts are crucial for the integrity of all tissues. Contrasting reports have been published about the role of Cdc42 in epithelial cell-cell contacts in vitro. In keratinocytes, it was suggested that Rac1 and not Cdc42 is crucial for the formation of mature epithelial junctions, based on dominant negative inhibition experiments. Deletion of the Cdc42 gene in keratinocytes in vivo slowly impaired the maintenance of cell-cell contacts by an increased degradation of β-catenin. Whether Cdc42 is required for the formation of mature junctions was not tested.We show now that Cdc42-deficient immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of β-catenin, but correlated to an impaired activation and localization of aPKCζ in the Cdc42-null keratinocytes. Inhibition of aPKCζ by the inhibitor Gö6983 reproduced the phenotype, suggesting that decreased activation of aPKCζ was sufficient to explain the defective junctional maturation. In the absence of Cdc42, Rac1 activation was strongly decreased, indicating that Cdc42 is upstream of Rac1 activation. These data reveal that Cdc42 is crucial for the formation of mature epithelial cell junctions between keratinocytes by regulating activation of aPKCζ.     

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.