【作者】 林显光；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理学， 2010， 博士

【摘要】 生物体的生命活动是在多方面因素的严格调控下进行的,囊泡的分泌过程在对生物体的调控作用中起着重要作用,是大多数宏观调控过程的分子基础。通过高分辨率电子显微镜观察发现,囊泡可以分为致密核心大囊泡(LDCVs)和突触小囊泡(SVs)两类。致密核心大囊泡中的内涵物主要是大分子蛋白质和神经肽类物质,突触小囊泡中的内涵物则主要是一些神经递质。而近年来随着电生理技术和细胞成像技术的迅猛发展,我们可以直接对活体细胞中囊泡的分泌事件进行高时间分辨率和高空间分辨率的观测,从而研究在这一过程功能基团(如蛋白质)之间的相互作用及其作用机理。UNC-31是由S.Brenner于1974年在筛选表型异常的EMS诱变的线虫时发现的。缺失UNC-31的线虫表现出运动共济失调(UNC)、反应迟钝、产卵障碍等表型。其哺乳动物中的同源物CAPS(Ca2+-dependent activator protein for secretion)于1992年在鼠脑中发现,已经证实其一种与囊泡分泌过程中不可缺少的重要蛋白,随即成为分泌研究领域中的热点对象之一。这一蛋白含有5个预测的功能结构域,从N端到C端,分别是动力蛋白激活蛋白1结合结构域(DBD)、C2结构域、PH结构域、Munc-13同源结构域以及致密核心大囊泡结合结构域(DCVBD)。一些侧面研究(如同源序列比对,竞争性过表达)表明这些结构域有着各自的功能,但是尚无直接的证据证明这些结构域确实在UNC-31行使其囊泡分泌相关功能中发挥作用或者仅仅是进化过程中的无实际作用的遗留片段。本文以此为切入点,将线虫作为模式生物,有机的结合了传统的线虫行为、药理学分析和近年来兴起的电生理技术、全内反射细胞成像技术以及活体荧光检测技术对这些结构域的功能进行了研究,这样就将微观上的分子或细胞水平上的观测结果和宏观上的表型联系起来。我们的结果表明：综合来看,这些功能结构域对于UNC-31行使分泌过程中的功能是缺一不可的,但Munc-13同源结构域却表现出一些特殊的性质。当线虫中的UNC-31缺少这一结构域时,其神经元细胞在电生理实验中分泌状态正常,类似于野生型；而其他实验中,如果线虫中UNC-31缺少这一结构域却都表现出类似于UNC-31基因缺失的突变体的表型。我们猜测这一异常的结果可能是由于电生理实验中必须的较高的基础钙浓度导致的特殊现象。更多还原

【Abstract】 Life activities of organisms is under strict control by a variety of regulation factors, in which the secretion of vesicle plays an important role and is the molecular basis of macro-control process. Observed by high resolution electron microscopy, vesicles can be divided into large dense core vesicles (LDCVs) and synaptic vesicles (SVs) categories. Large dense core vesicles mainly contain macromolecular protein and neuropeptides, and synaptic vesicles mainly carry neurotransmitter. With the rapid development of electrophysiological techniques and cellular imaging technology in recent years, we can directly monitor secretory vesicles in the high time resolution and high spatial resolution on the living cells, and to study the functional groups (such as proteins) interaction in the process and its mechanism.UNC-31 is discovered by S.Brenner in 1974 in the screening of EMS induced nematode mutation. Lack of UNC-31 in C. elegans leads to uncoordinated movement (UNC), unresponsive to stimulation, and defect in spawning. The mammalian homolog of CAPS (Ca2+-dependent activator protein for secretion) was found in 1992 in rat brain and has been confirmed one of indispensable protein in the process of vesicle secretion. Then It soon became a hot spot in filed of secretion study. This protein contains five predicted functional domains, from N-terminus to C terminus, they are dynactin 1 binding domain (DBD), C2 domain, PH domain, Munc-13 homology domain, and large dense core vesicle binding domain (DCVBD). Some oblique studies (such as sequence alignment, competitive overexpression) showed that these domains have their own features, but there is no direct evidence that these domains play roles in the UNC-31 functions in vesicle secretion process or only the none sense meaningless legacy of evolution. This thesis selects C. elegans as model organism, and combined of the traditional worm behavior and pharmacological assay, recently developed electrophysiological technique, total internal reflection fluorescence microscopy technique and in vivo fluorescence imaging to detect function of these domains. Our results demonstrate that these domains are all indispensible for the function of UNC-31 in exocytosis process, but the Munc-13 homology domain has shown some special properties. UNC-31 without this domain can still works in electrophysiological experiments, the neuron cells of C.elegans express a normal secretion, similar to wild type. In other experiments if knock out this domain from UNC-31 the worm shown phenotypes similar to the UNC-31 gene deletion mutants. We suspect that this special phenomenon may be due to extra high basis calcium concentration in electrophysiological experiments.更多还原