【作者】 徐平勇；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理， 2004， 博士

【摘要】 胰岛素是机体最重要的激素之一, 它调节机体的血糖稳定、促进细胞代谢、调节细胞的分裂分化和生长发育。胰岛素储存在致密核心大囊泡中,通过囊泡分泌释放到细胞外。胰岛素的释放需要经过囊泡转运、锚定、启动和融合等多个步骤。Syntaxin1A (Stx1A)和 Munc18a在囊泡的转运和细胞分泌中发挥着关键的调控作用。然而它们的调控机制尚有待于进一步的研究。 本论文首先用Confocol和宽场荧光显微镜技术研究了在胰岛素分泌细胞系INS-1以及非分泌细胞系中国仓鼠卵巢细胞(CHO)中Stx1A和Munc18a的胞内定位和相互作用。结果表明在INS-1细胞膜上Munc18a与Stx1A呈簇状共定位分布,但是不与Stx1A的氨基端Habc结构域缺失突变体共定位,表明Stx1A的Habc结构域在Stx1A与Munc18a相互作用以及Munc18a在膜上定位中起着至关重要的作用。在没有内源性Stx1A表达的CHO细胞中,EGFP标记的Stx1A不能定位到细胞膜上,即使与Munc18a共表达也不能定位到细胞膜上,表明Munc18a不足以将Stx1A转运到细胞膜。相反地,在INS-1细胞中Stx1A的上膜有助于Munc18a向细胞质膜的转运。Stx1A的一种双点突变体(Stx1A-L165A/E166A)可以使它保持开放的构型,在体外结合实验发现不与Munc18a发生相互作用,但是我们发现在体内仍能与Munc18a呈簇状共定位在细胞膜上,表明Munc18a可以通过结合闭合构型或者开放构型的Stx1A而转运到细胞膜上。体内荧光共振能量转移(FRET)证明了共定位的Munc18a与Stx1A发生了相互作用,但是Stx1A-DM尽管与Munc18a共定位分布,却没有观察到明显的FRET的发生。表明Munc18a与Stx1A以及Stx1A-DM之间可能具有不同的作用方式。 最后,用西伯利亚森林脑炎病毒(SFV)表达系统在原代β细胞中过量表达Munc18a研究其对胰岛素分泌的影响,通过两次连续钙离子光解释放(flash)技术发现过量表达Munc18a的细胞与对照细胞相比,第一次flash的慢簇发成分(SlowlyReleasable Pool,SRP )和延迟成分(sustained component)的大小减少了50 %,但 I<WP=6>是快簇发成分(Rapidly Releasable Pool,RRP)的大小却没有什么改变。而紧接着的第二次flash的RRP,SRP以及延迟成分都降低了大约50%。但是过量表达Munc18a对RRP或者SRP的分泌动力学特性都没有明显的改变。表明过量表达Munc18a由于稳定了Stx1A的闭合构型,阻止了囊泡与质膜之间反式SNARE复合物的形成,因而抑制了囊泡向RRP的再填充(refilling)过程,进而抑制了胰腺β 细胞的分泌。另外,我们还发现过量表达闭合构型Stx1A或者开放构型的Stx1A对胰岛素的分泌没有什么影响,表明在胰岛素的分泌调控中Stx1A由闭合构型转换为开放构型不是分泌的限速步骤。更多还原

【Abstract】 Insulin is one of the most important versatile hormones, and its functions includeregulation of glycemia homeostasis, enhancement of anabolism, control of cell divisionand differentiation, and modulation of cell growth and development. Insulin is stored inlarge dense core vesicles and released by exocytosis, a multistage process involvingt-ransport of vesicles to the membrane, their docking, priming and final fusion with theplasma membrane. Syntaxin 1A (Stx1A) and Munc18a play essential roles in vesiculartrafficking and exocytosis. The molecular mechanism and the functional roles of theirinteraction remain to be explored. In the present study, we have studied the intracellularlocalization and interaction of Stx1A and Munc18a in insulin secreting INS-1 cells as wellas non-secretary CHO cells by confocal and evanescent field fluorescence imaging. Wefound that Munc18a colocalized with clusters of Stx1A and its open-form mutant, but notthe mutant lacking Habc domain, at the plasma membrane in live INS-1 cells, suggestingthe interaction of Munc18a with the Habc domain of Stx1A is necessary for thetranslocation of Munc18a to the plasma membrane. In CHO cells, where no endogenousStx1A is reported, Stx1A failed to localize to the plasma membrane even in the presenceof coexpressed Munc18a, suggesting Munc18a is not required for the transportation ofStx1A to the plasma membrane. On the other hand, Munc18a, which displayed a diffusedcytoplasmic localization alone in INS-1 cells, was translocated to the plasma membranewhen coexpressed with Stx1A, suggesting that the trafficking of Stx1A drives Munc18a tothe functional sites. Interestingly, Stx1A-L165A/E166A (Stx1A-DM), an openconfiguration of Stx1A that does not interact with Munc18a in a pull down assay, alsoexisted in clusters on the plasma membrane and Munc18a was concentrated on the samemicrodomains of Stx1A-DM, suggesting either Munc18a is recruited to the microdomainsthrough binding to the open-form Stx1A-DM. in vivo FRET measurement demonstratedthe interaction between Munc18a and Stx1A, whereas no significant FRET signal was III<WP=8>observed for Munc18a and the open-form Stx1A mutant despite their colocalization,implying the different ways of interaction between Stx1A and Stx1A-DM with Munc18a. By overexpression of Munc18a in primary cultured pancreatic β cells, we have shownthat in response to the first flash, the slow burst component was inhibited as well as thesustained component of exocytosis, whereas the fast burst component remains unaltered.This effect is more obvious when the responses to the second flash were compared, wherethe recovery of both the fast burst and the slow burst component was blocked. The resultsfavor a role for Munc18a to block the formation of trans-SNARE complexes between thevesicle and the plasma membrane by stabilizing Syntaxin in closed configuration, henceinhibiting the recruitment of vesicle to the release-ready pools. Additionally, we havefound that overexpression of neither the wild type Stx1A nor the open-form Stx1A affectsexocytosis, suggesting the availability of Stx1A in its open form is not a rate limiting stepin insulin secretion.更多还原