【作者】 王亮；

【导师】 潘俊敏；

【作者基本信息】 清华大学 ， 生物学， 2013， 博士

【摘要】 真核生物的纤毛（或鞭毛）是一种由“9+2”微管结构组成的细胞表面突出的保守的细胞结构。纤毛在哺乳动物发育、细胞运动、细胞周期调控和信号转导等方面起着重要的作用。纤毛的结构或者功能上的缺失，与多种人类疾病相关，因此近年来关于纤毛的研究一直是个热点。而纤毛的组装与解聚对纤毛的结构与功能是关键的，但是具体机制还了解的不多。纤毛轴丝的微管结构成分来源于胞质中的前体物质库（precursor pool），但是该前体物质库是如何调控的却了解很少。本文利用单细胞衣藻作为模式生物来进行研究鞭毛组装与解聚的分子机理。在之前的研究中发现了一种在鞭毛再生过程中发生磷酸化的蛋白质CrKinesin13/CrKin13，是细胞微管解聚马达蛋白家族中的一员，CrKin13可以解聚细胞质微管骨架以提供鞭毛再生之所需的游离微管蛋白，对于衣藻鞭毛的再生至关重要。本文继续探究了CrKin13在衣藻鞭毛组装与解聚过程中所起的功能和具体机制。本文进一步验证了在细胞鞭毛再生初期确实存在细胞质微管骨架的解聚现象。利用突变体及药物处理研究发现细胞质微管骨架解聚发生在掉鞭毛之前，只要CrKin13发生磷酸化，细胞质微管就发生解聚，与鞭毛脱落与否无关。在CrKin13磷酸化调控机制研究中，磷酸化和非磷酸化形式的CrKin13均可在体外依赖ATP解聚微管，但是磷酸化形式的CrKin13解聚微管的活性降低。通过质谱分析，鉴定出CrKin13的主要磷酸化位点为S100，次要磷酸化位点为S522和T469，并且S100磷酸化位点的磷酸化对于CrKin13定位于胞质微管骨架结构上是至关重要的。而在鞭毛再生过程中，鞭毛中的CrKin13有一个富集的过程，也可能参与到鞭毛内微管的动态学变化过程。本文还初步探究了CrKin13的磷酸化激酶和相互作用蛋白，通过酵母双杂交的方法初步筛选到了相互作用蛋白TOG1。而在鞭毛缩短时CrKin13鞭毛运输的机制方面，发现了N端对于CrKin13进入鞭毛是必须的，而M端、C端可能共同调节CrKin13进入鞭毛的信号。因此，磷酸化CrKin13解聚胞内微管以调节前体物质库，提供衣藻细胞再生鞭毛所用的游离微管蛋白，在鞭毛组装过程中发挥重要的作用。更多还原

【Abstract】 Cilia (or flagella) in eukaryotes are highly conserved organelles and "9+2" microtubule based structures, protruding from the cell body. Cilia play animportant role in mammalian development, cell motility, cell cycle regulationand signal transduction. In mammalian cells, defects in cilia structure orfunction lead to varieties of human diseases, so recently many researchers focuson this area. Assembly and disassembly of cilla play a key role in cilia structureand function, but little is known about the mechanism of cilla assembly anddisassembly. Microtubule based cilia come from the cytoplasmic “precursorpool”, but little is understood about the regulation of cytoplasmic pool.Here, we use unicellular algae chlamydomonas as model organism forresearch on the molecular mechanism of flagellar assembly and disassembly.Studies have shown that a protein is phosphorylated during flagellarregeneration, named CrKinesin13/CrKin13, which belongs to microtubuledepolymerizing kinesin family. It is crucial for flagellar regeneration, becausethat it depolymerizes cytoplasmic microtubules to provide free tubulins forflagellar regeneration.In this paper, we further explore the mechanism of CrKin13on flagellarregeneration and shortening. Based on the previous studies, we further verifythat cytoplasmic microtubules are depolymerized at the early stage of flagellarregeneration. Using mutants and drug treatments, it is also found thatcytoplasmic microtubule depolymerization occurs before the flagellar loss, andcytoplasmic microtubules are depolymerized as long as CrKin13isphosphorylated, which is not related with flagella loss per se. Moreover, in vitroassay shows that both of phosphorylated and non-phosphorylated forms ofCrKin13possess ATP-dependent microtubule depolymerizing activity, but theactivity of phosphorylated form of CrKin13is lower. The phosphorylation ofCrKin13occurs at residues S100as primary site and S522, T469as secondarysites by mass spectrometry. Mutation of CrKin13phosphorylation site at S100but not at other residues prevents CrKin13targeting to cytoplasmicmicrotubules, which explains the phosphorylation of CrKin13at the early stage of flagellar regeneration. While the enrichment of CrKin13in the regeneratingflagella might be involved in the flagellar microtubule dynamics.Additionally, the kinase responsible for CrKin13’s phosphorylation and itspartner are also examined. TOG1screened as candidate by yeast two hybridmethod need to be further analyzed. To reveal the mechanism of targeting toshortening flagella, our results find that N-domain is necessary for flagellartargeting and M-, C-domain may be co-regulate the CrKin13targeting signals.Thus, we propose that CrKin13is regulated by its phosphorylation todepolymerize cytoplasmic microtubules to provide tubulin precursors duringflagellar regeneration, so it plays a key role in flagellar regeneration.更多还原