BIOGENESIS OF THE GOLGI APPARATUS

描述（由申请人提供）：了解在发育、细胞分裂和细胞应激过程中建立和维持高尔基体的分子机制是生物医学研究的重要目标。我们的长期目标是鉴定高尔基结构所需的蛋白质并阐明其机制。这项工作是通过基于sirna的筛选候选物，特别是高尔基蛋白，以及基于细胞的高尔基组装测定，然后结合生化、渗透细胞、结构和计算分析进行敲除后的修复。我们的筛选确定了高尔基蛋白160 （G160）是高尔基膜向内运动的关键要求，并表明它的缺失会阻止分散的高尔基蛋白组装，从而损害伤口愈合过程中的定向分泌和细胞迁移。这一观察结果提出了一种可能性，我们可以回答有关运动受体复合物的未知身份和调节的基本问题，运动受体复合物沿着微管向内移动分泌货物和高尔基膜。为了验证G160招募或激活动力蛋白运动复合物的假设，我们将1)识别并功能表征其与运动细胞质动力蛋白的相互作用，2)识别并功能表征其与高尔基体膜的arf1依赖性相互作用，3)通过开发一种技术来测试高尔基体对G160基于运动的急性依赖性，该技术使用光在几秒的时间范围内灭活G160。4)阐明细胞分裂过程中高尔基体扩散和遗传的调控机制。这些目标得到了关键初步调查结果的支持。高尔基膜的微管+尖端捕获需要G160。G160 c项直接与动力蛋白复合物的中间链结合。G160是动力蛋白高尔基缔合所必需的，也足以用于功能性运动募集，因为G160靶向线粒体募集动力蛋白并诱导线粒体向内运动。G160 n项介导其依赖arf1的膜结合，其结合受到调节，从而使G160循环到细胞外周并返回到微管上。G160的光失活表明高尔基体严重依赖G160为基础的向内运动，因为早期的高尔基酶不断循环到细胞周围。最后，G160在有丝分裂时与高尔基体分离，但仍与动力蛋白结合并在纺锤极收集。因此，我们的实验准备揭示长期寻找的动力蛋白运动的高尔基受体成分，并阐明从微管运动中解耦高尔基膜以允许高尔基分解为子细胞的调节。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms that establish and maintain the Golgi during development, cell division, and cellular stress are significant ongoing goals in biomedical research. Our long-term goal is to identify proteins required for Golgi structure and elucidate their mechanism. This work was initiated using an siRNA-based screen targeting candidates, especially golgins, and cell-based Golgi assembly assays followed by rescue after knockdown in conjunction with biochemical, permeabilized-cell, structural, and computational assays. Our screen identified golgin-160 (G160) as a critical requirement for inward motility of Golgi membranes and showed that its absence arrests Golgi assembly at the dispersed ministack stage impairing directed secretion and cell migration during wound healing. This observation raised the possibility that we could answer fundamental questions concerning the unknown identity and regulation of the motor receptor complex that moves secretory cargo and Golgi membranes inward along microtubules. To test the hypothesis that G160 recruits or activates the dynein motor complex we will 1) identify and functionally characterize its interaction with the motor cytoplasmic dynein, 2) identify and functionally characterize its Arf1-dependent interaction with the Golgi membrane, 3) test the acute dependence of the Golgi on G160-based motility by developing a technique to inactivate G160 using light in a time scale of seconds, and 4) elucidate the control mechanism that allows Golgi dispersal and inheritance during cell division. These aims are supported by key preliminary findings. G160 was required for microtubule +tip capture of Golgi membranes. The G160 C-term bound directly to the intermediate chain of the dynein complex. G160 was required for dynein Golgi association and it was also sufficient for functional motor recruitment as G160 targeted to mitochondria recruited dynein and induced mitochondrial inward motility. The G160 N-term mediated its Arf1-dependent membrane association and its binding was regulated such that G160 cycled to the cell periphery and returned inward on microtubules. Photo-inactivation of G160 showed that the Golgi acutely depends on G160-based inward motility because early Golgi enzymes constantly cycle to the cell periphery. Finally, G160 dissociated from the Golgi at mitosis but remained bound to dynein and collected at spindle poles. Thus, our experiments are poised to uncover components in the long-sought Golgi receptor for the dynein motor and elucidate the regulation that uncouples Golgi membranes from microtubule-based motility to allow Golgi partitioning into daughter cells.