【作者】 戴洁琼；

【导师】 杨卫军；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 卤虫是一种生活在盐田、盐湖等高盐环境下的小型甲壳动物。为了抵抗外界极端环境的胁迫,它们进化出一种非常特殊的生殖模式,即在适宜的环境条件下,通过卵胎生方式直接产生无节幼体,而在不适宜的环境下,通过卵生方式产生休眠胚胎。这些休眠胚胎的发育停滞于原肠胚期,内部仅维持极低的代谢水平,直至休眠状态被打破。在休眠胚胎的孵化发育过程中,细胞内部发生了剧烈的变化,包括能量代谢、RNA和蛋白质的合成、细胞分化与形态发生等,以完成个体由原肠胚向前无节幼体的转变过程。然而在这一极其复杂的生理过程中,细胞分裂乃至DNA复制都是完全停止的,此时整个胚胎的细胞数始终维持在4000左右。尽管科学家们普遍认为,卤虫休眠胚胎发育过程中出现的这种细胞分化及形态发生完全独立于细胞分裂的现象对于维持胚胎的稳定性和存活能力具有重要的意义,但这一过程的相关分子机制仍然未知。本研究主要围绕卤虫休眠胚胎发育过程中p90核糖体S6蛋白激酶(RSK)途径调控细胞周期的分子机制而展开。RSK作为一种调控丝裂原活化蛋白激酶(MAPK)信号途径下游分子活性的丝氨酸、苏氨酸激酶,广泛参与了诸如转录调控、细胞周期调节、细胞存活等细胞内大量生理活动的调节过程。本研究首先分析了RSK分子在卤虫胚胎和幼虫发育过程中的表达和活性,又通过一系列体内蛋白活性抑制实验确认了RSK激酶活性对于细胞周期调控和有丝分裂发生的作用。在此基础上,我们又针对卤虫RSK的分子结构与功能活性,以及卤虫RSK途径的上下游作用分子等进行了鉴定研究。此外,我们利用高等动物细胞系和爪蟾系统等表达卤虫RSK分子,由此比较分析了卤虫RSK与其它物种RSK的结构功能区别。研究结果显示,RSK分子在卤虫各个生长发育时期均有表达,但仅在休眠胚胎发育后期的前无节幼体出壳阶段才被磷酸化激活。此时,RSK的活化过程恰与胚胎细胞恢复有丝分裂的过程同时发生。一旦在卤虫胚胎发育过程中抑制RSK活性,幼体就会出现严重的生理缺陷,表现为胸腹部区域出现大量组织空腔。利用BrdU对分裂细胞进行标记和检测的结果证实了这些缺陷部位的细胞有丝分裂活性明显受到抑制。在此作用途径中,RSK激酶的活化伴随着上游ERK激酶的活化而发生,并且卤虫RSK分子中含有ERK结合序列的C末端区域对于激酶活化至关重要。在此途径的下游,RSK可能与卤虫休眠胚胎特异性表达的小分子热休克蛋白p26存在一定的相互关系。此外,在爪蟾卵母细胞的成熟过程中,卤虫RSK分子能够诱导卵母细胞提前发生生发泡破裂现象,但无法促使其完成整个成熟过程,致使卵母细胞长时间停滞在初始阶段,出现黏化、干瘪等现象。本研究由此表明,RSK途径在卤虫休眠胚胎发育过程中参与了G2/M细胞周期停滞的终止以及有丝分裂发生等调控过程,这对于幼虫的生长至关重要。此结果不但为进一步揭示卤虫休眠胚胎特殊发育模式的分子机制奠定了理论基础,而且还扩展了RSK这一重要蛋白激酶的功能新领域。更多还原

【Abstract】 Artemia is a genius of Crustacean widely distributing in salterns and salt lakes. It possesses powerful adaptations to extreme environments. As a strategy to cope with environmental stresses, Artemia has evolved a special reproductive mode in that in addition to giving birth to nauplii by the ovoviviparous pathway, this genus also releases encysted embryos by the oviparous pathway. Released encysted embryos are developmentally arrested at the gastrula stage with a low metabolic rate and complete turnoff of replication, transcription, and translation; however, without loss of embryonic viability. During the development of Artemia-encysted embryos, no cell division or DNA synthesis occurs before emergence, and the number of nuclei remains at about 4000 per embryo despite the great number of internal events including deposited energy mobilization, RNA and protein synthesis restoration, cellular differentiation, and associated morphological changes required for the development from gastrula to prenauplius.The mechanism of this unusual developmental pattern is still unclear. It is considered that the absence of cell division during the early embryonic development of Artemia is an adaptation to environmental threats, which confer on embryos their amazing stability and viability.Our research focuses on the molecular mechanism of p90RSK pathway in cell cycle regulation during development of Artemia-encysted embryos. RSK is a family of serine/threonine kinases that mediate signal transduction downstream of mitogen-activated protein kinase cascades. RSK family members have been reported to be multifunctional in the regulation of diverse cellular processes including transcriptional regulation, cell cycle control, cell survival, and many others. In the present study, we analyzed the expression and activation patterns of Artemia RSK, and identified its function to the regulation of cell cycle arrest and mitogenesis through in vivo knockdowns, during the embryonic and larval development of Artemia. Basing on this, the structure-functional studies of Artemia RSK, and the exploring of its upstream and downstream molecules were carried out. Besides, the functional analysis and comparison between Artemia RSK and other family members was performed in transgenic cell lines and Xenopus.Our results represent that, Artemia RSK was established to be specifically activated at the very beginning of emergence and was coupled with mitogenesis during the post-embryonic and early larval developmental stages. In vivo knockdown of RSK activity consistently induced abnormal individuals with distinct gaps between the exoskeleton and the internal tissues in the developing thoracic and abdominal regions. By BrdU labeling and mitotic index analysis, mitoses were detected to be largely inhibited in those affected segments. In this pathway, RSK was activated concomitantly with ERK activation, and the C terminal of Artemia RSK containing an ERK docking sequence was crucial to the kinase activation. In the downstream regulation, there might be an intracellular connection between RSK and the molecular chaperone p26. Otherwise, expression of Artemia RSK in Xenopus oocytes with endogenous RSK inhibition could induce an advance of showing GVBD (germinal vesicle breakdown) evidence; however, they did not proceed to a distinct GVBD subsequently.Our research, thus, indicates that the RSK pathway is essential in the post-embryonic and early-larval development of Artemia by playing a major role in the termination of cell cycle (G2/M phase) arrest and the promotion of mitogenesis, essential for development. These findings not only provide insights into the molecular mechanism regulating the special developmental pattern of Artemia-encysted embryos, but also reveal further aspects of RSK functions.更多还原