【作者】 周焰；

【导师】 李和；

【作者基本信息】 华中科技大学 ， 人体解剖与组织胚胎学， 2019， 硕士

【摘要】 钙反应性反式激活子(calcium-responsive transactivator,CREST)是神经发育过程中分裂后神经元内高水平表达的一种核蛋白,通过调节钙依赖性的基因转录影响神经元树突的发育,参与维持成熟神经元的形态。缺乏CREST的基因敲除小鼠表现出明显的运动障碍、树突生长异常和分支减少。我们实验室的前期研究发现,无血清培养基诱导小鼠成神经瘤母细胞(N2a细胞)分化过程中CREST表达增加,并呈颗粒状分布于细胞质中,过表达CREST可通过激活神经元分化相关基因的转录,进而促进神经突起的生长和神经元的分化。神经元分化建立在神经突起(轴突、树突)的生长和分支的基础上。神经突起内部微管的组装和延伸是神经突起生长的骨架支撑,微管发育的缺陷会引起神经系统形态和功能异常。神经突起的形成和生长依赖于多种微管相互作用蛋白对微管骨架稳定性和动态性的协同调节,它们通过与微管骨架结合,调节微管的组装、稳定、解聚或断裂。CREST在神经元分化过程中出现的细胞质颗粒状分布现象提示其可能存在着胞质功能,通过对微管骨架的直接影响调节神经突起的生长。本研究利用免疫荧光和免疫共沉淀实验分析了CREST与α-微管蛋白(α-tubulin)的定位和结合关系,以及二者相互作用的分子区域;通过对微管骨架的形态学观察,对微管稳定性的检测,以及对微管正末端组装的动态观察,深入解析了CREST通过调节微管的稳定性和动态性促进神经突起生长的机制。1.CREST通过其MFD结构域与α-tubulin的中间区域相互作用为了明确在分化的N2a细胞中CREST在细胞质中的颗粒状分布是否与微管相关,我们首先通过免疫荧光双标检测内源性CREST与α-tubulin的分布,结果显示:在未分化的N2a细胞中,CREST主要分布于细胞核内,α-tubulin在细胞质中分布较为弥散,组装状态(纤维状)的微管较少,细胞质中仅有少量CREST颗粒与纤维状微管共定位;而在无血清诱导分化的N2a细胞中,α-tubulin大量组装成纤维状微管,CREST在细胞质中的免疫反应性显著增强,呈颗粒状与微管骨架形成良好的共定位分布关系。通过免疫共沉淀分析显示:在出生后一周的小鼠大脑皮质和无血清诱导分化的N2a细胞中,内源性CREST与α-tubulin均存在相互作用。利用荧光蛋白GFP和mCherry作为标签,分别构建pEGFPCREST、pmCherry-tubulin及二者的结构域缺失突变质粒,经免疫共沉淀检测发现:CREST通过其多功能结构域MFD(246-317aa)与α-tubulin的中间段(132-227aa)结合。2.CREST增加微管的稳定性为了明确CREST与微管的结合是否对微管的稳定性产生影响,我们首先通过免疫荧光对N2a细胞神经突起生长过程中微管的组装和纤维化现象进行检测:无血清培养诱导分化或过表达CREST的N2a细胞中,α-tubulin大量聚集成纤维状的微管束,而未分化及转染对照质粒的N2a细胞中α-tubulin主要呈弥散状分布于胞质中,少见纤维状的微管束形成。通过蛋白免疫印迹对α-tubulin的乙酰化和去酪氨酸修饰水平(稳定微管的标记)进行检测显示:无血清诱导分化或过表达CREST均能增加N2a细胞中α-tubulin的乙酰化和去酪氨酸化修饰,而无血清培养诱导N2a细胞分化的同时沉默CREST,α-tubulin的以上两种修饰水平显著降低。以上结果说明,在神经突起的生长过程中,CREST可能作为一种微管相关蛋白(microtubule-associated protein,MAP)通过与α-tubulin相互作用增加微管的稳定性。3.CREST增加微管的动态组装神经细胞突起的生长依赖于微管稳定性和动态组装的协调。完全稳定的微管并不利于突起生长,而稳定微管突起端的动态组装才是突起伸长的关键。为了动态观察CREST对微管末端动态性的影响,我们构建可示踪微管末端动态性的GFP-EB1质粒,将其转染N2a细胞后,通过激光共聚焦显微镜追踪GFP-EB1荧光点的数量和移动速度,记录分析神经突起中微管的生长情况。结果显示:在无血清诱导分化或过表达CREST的N2a细胞中,GFP-EB1荧光点的数量显著增多,运动速度明显加快,说明处于生长状态的微管数量增多,微管生长速度增快;在无血清诱导分化的N2a细胞中过表达CREST,其突起中处于生长状态的微管数量增多,微管生长速度增快;而无血清诱导分化的N2a细胞中沉默CREST则导致突起中处于生长状态的微管数量显著减少、末端生长速度显著降低。以上结果说明,CREST能够促进微管正末端的动态组装。结论:CREST是一种微管相关蛋白,在神经元分化的过程中呈颗粒状定位于细胞质,通过自身的MFD结构域与α-tubulin的中间段相互作用;CREST通过与α-tubulin的结合增加微管的稳定性,并促进神经突起中微管正末端的动态组装,进而促进神经突起的生长。更多还原

【Abstract】 The calcium-responsive transactivator(CREST)is a nuclear protein highly expressed in post-mitotic neurons during neurodevelopment.It affects the development of neuronal dendrites by regulating calcium-dependent gene transcription,and involved in maintaining the morphology of mature neurons.CREST-knockout mice showed significant dyskinesia,abnormal dendritic growth and reduced branching.Previous studies in our laboratory found that the expression of CREST increased during the differentiation of mouse neuroblastoma cells(N2a cells)induced by serum-free medium,and distributed in the cytoplasm in a granular manner.Overexpression of CREST can promote neurite growth and neuronal differentiation by activating the transcription of genes related to neuronal differentiation.Neuronal differentiation is based on the growth and branching of neurites.The assembly and extension of microtubules in neurites is the skeleton support of neurite growth.The defects of microtubule development will cause abnormal morphology and function of nervous system.The formation and growth of neurites depend on the synergistic regulation of the stability and dynamics of microtubule skeletons by various microtubule interacting proteins,they regulate the assembly,stability,depolymerization or rupture of microtubules by binding to microtubule skeletons.The granular distribution of cytoplasm in the process of neuronal differentiation suggests that CREST may have cytoplasmic function and regulate the growth of neurites through the direct influence of microtubule cytoskeleton.In this study,the interaction characteristics between CREST and α-tubulin as well as their molecular regions of interaction were analyzed by immunofluorescence and immunoprecipitation experiments.Through the morphological observation of the microtubule skeleton,the detection of the stability of the microtubule and the dynamic observation of the plus end assembly of the microtubule,the molecular mechanism of CREST promoting the neurite growth by regulating the stability and dynamics of the microtubule was deeply analyzed.CREST interacts with α-tubulin middle region through its MFDIn order to determine whether the granular distribution of CREST in cytoplasm is related to microtubules,the distribution of endogenous CREST and α-tubulin in N2 a cells was detected by immunofluorescence double labeling.The results showed that in undifferentiated N2 a cells,CREST was mainly distributed in the nucleus,α-tubulin was dispersed in the cytoplasm,there were fewer assembled(fibrous)microtubules,and only a small number of CREST particles were co-located with fibrous microtubules in the cytoplasm.In serum-free differentiated N2 a cells,a large number of α-tubulin were assembled into fibrous microtubules,and the immunoreactivity of CREST in cytoplasm was significantly enhanced,showing a good co-localization and distribution relationship between granular and microtubule skeleton.Immunoprecipitation analysis showed that endogenous CREST and α-tubulin interacted in the cerebral cortex of mice one week after birth and in serum-free differentiated N2 a cells.In order to further clarify the molecular region of the interaction between CREST and α-tubulin,we used GFP and mCherry fluorescent protein as tags to construct the full-length of pEGFP-CREST and mCherry-tubulin and their respective structural deletion mutants,respectively.The patients were divided into groups for immunoprecipitation test,The results showed that CREST binds to the intermediate segment of α-tubulin(132-227aa)through its multifunctional domain MFD(246-317aa).CREST increases the stability of microtubulesIn order to determine whether the binding of CREST to microtubules has an effect on the stability of microtubules,we first detected the assembly and fibrosis of microtubules during neurite growth of N2 a cells by immunofluorescence.The results showed that α-tubulin aggregated into fibrous microtubule bundles in N2 a cells induced by serum-free culture or overexpressed CREST,while the formation of fibrous microtubule bundles is rare in cytoplasm in N2 a cells which were not differentiated or transfected with control plasmids.The acetylation and detyrosine modification levels of α-tubulin(labeling of stable microtubules)were detected by Western blot.The results showed that serum-free differentiation or overexpression of CREST could increase the acetylation and detyrosine modification of α-tubulin in N2 a cells.However,the above two modification levels of α-tubulin were significantly decreased in CREST silenced cells.These results suggest that CREST increases the stability of microtubules by interacting with α-tubulin during neurite growth.CREST is a new microtubule-associated protein(MAP).CREST increases dynamic assembly of microtubulesThe growth of neurites depend on the close coordination of microtubule stability and dynamics.A completely stable microtubule is not conducive to protuberance growth,and the dynamic assembly of the plus end of the stable microtubule skeleton is the key to neurites elongation.In order to dynamically observe the effect of CREST on the dynamics of microtubule end,we constructed a GFP-EB1 plasmid which can trace the dynamics of microtubule end.After transfected into N2 a cells,the number and movement speed of GFP-EB1 fluorescence points were tracked by laser confocal microscope.The growth of microtubules in neurites was recorded and analyzed.the results showed that in N2 a cells without serum-induced differentiation or overexpression of CREST,the number of GFP-EB1 fluorescent spots was significantly increased and the movement speed was significantly accelerated,indicating that the number of growing microtubules as well as the growth rate of microtubules was increased.Overexpress CREST in serum-free differentiated N2 a cells,the number of growing microtubules and the growth rate of microtubules in the neurites were both increased.However,the silencing of CREST in serum-free differentiated N2 a cells resulted in a significant decrease in the number of growing microtubules in the neurite and a significant decrease in the rate of terminal growth.These results suggest that CREST can promote the dynamic assembly of the microtubule.Conclusion: CREST is a new microtubule associated protein,which is located in the cytoplasm in the process of neuronal differentiation and interacts with the intermediate segment of α-tubulin through its MFD domain.CREST increases the stability of microtubules by combining with α-tubulin,and promotes the dynamic assembly at the plus end of microtubules in neurites,thus promoting the growth of neurites.更多还原