果蝇中ER-Golgi界面的遗传、细胞和分子表征

The endoplasmic reticulum (ER) is the largest, most versatile organelle in eukaryotic cells, capable of generating other organelles and subcellular compartments and establishing coordinated relations with them. In the early secretory pathway, cargo destined to be secreted is collected at specialized regions of the ER called ER exit sites (ERES). From ERES, cargoes are transferred to the Golgi into membrane vesicles. Vesicles budding from the ER are generated by the coat protein II (COPII) complex, highly conserved in all eukaryotes. Structural studies have shown that budding of COPII vesicles from ERES is mediated by the assembly of a cage of 60–90 nm in diameter. In animals, despite this, many secreted proteins exceed the dimensions of this cage and yet they are efficiently secreted, raising the question of how this happens and whether specific mechanisms have evolved in animals to deal with newly evolved matrix proteins such as collagens. Furthermore, the topology of the ER-Golgi interface and the identity of carriers there allowing secretion of large proteins is not known. While many components have been found through genetic screenings that affect ER exit of cargo, few are known to affect post-ER traffic, indicating that ER-to-Golgi cargo transfer at the ER-Golgi interface is the critical and most regulated step in secretion. Due to the high concentration of proteins on this cytoplasmic interface and the dynamic behavior required to regulate traffic, it has been postulated that proteins at the ER-Golgi interface possess the properties of a membrane‐less liquid droplet. TANGO1, a human ERES-localized transmembrane protein, has been proposed to function as a Collagen receptor ensuring coupling of cargo to the COPII coat. However, my research has shown that Drosophila Tango1 is required for secretion of Collagen, but also of all other cargoes. Moreover, Tango1 is an essential factor in the morphogenesis of ERES and the maintenance of the ER-Golgi interface. The powerful genetic tools available in Drosophila, coupled to evolutionary conservation and little genetic redundancy, offer an opportunity to investigate the organization of the ER-Golgi interface. I hypothesize that close proximity or direct contact of ER and Golgi membrane compartments mediated by Tango1 imposes structural and functional coupling characteristic of metazoan ER exit sites, allowing efficient transport from ER to Golgi. To test this main hypothesis, my research will (1) characterize in detail the function of Tango1 and its lumenal and cytoplasmic parts, (2) image the ER-Golgi interface at high resolution using state-of-the-art light and electron microscopy imaging techniques (SIM, APEX-TEM, FIB-SEM) and (3) screen through a proximity labeling by APEX proteomic approach proteins that localize and function at the ER-Golgi interface. In the course of this project, I expect to obtain fundamental insights both general about ER organelle communication and interfacing, and specific about protein secretion. These insights will help illuminate basic areas that remain controversial or poorly understood, such as the transport of large proteins, the dynamic coordination and physical segregation of anterograde and retrograde traffic, the specificity of cargo adaptors, the evolution of the metazoan secretory pathway and the nature of the ER-Golgi interface. Given the conservation of Tango1 and the relevance of the extracellular matrix and the secretory pathway to human health, insights obtained in the course of this project may have broad implications for the understanding and treatment of diseases involving deficits in secretion of Collagen and other proteins, including congenital syndromes. Finally, the experiments in this proposal will characterize the nature of the ER-Golgi interface, which will help better understand inter-organelle communication and formation of organelle contact sites and interfaces, particularly those formed by the ER.

ER能产生多种其他细胞器和亚细胞隔室，并与它们建立协调关系。分泌性蛋白在ERES被装载到COPII囊泡中并运输至Golgi。超出COPII大小的大蛋白如何被有效分泌以及是否有特定机制来处理大蛋白存在争议。ER-Golgi界面的拓扑结构和ER-Golgi间是否有大蛋白特殊载体未知。我们发现果蝇Tango1是ERES形态发生和ER-Golgi界面维持的重要因素。我假定，由Tango1介导的ER和Golgi膜隔室的紧密接近或直接接触影响ERES结构和功能的偶联，货物分子因此有效的从ER被运输到Golgi中。本项目将：（1）详细表征Tango1及其结构域的功能；（2）对ER-Golgi界面进行高分辨率成像；（3）筛选定位及作用于ER-Golgi界面的蛋白。我期望项目能阐明某些问题，如大蛋白的运输、货物分子相应受体的特异性、ER-Golgi界面本质等，更好理解细胞器间互作和细胞器接触位点和界面形成。

运往高尔基体前，在ER出口站点(ERES)收集分泌货物。数十年的研究提供了ER-高尔基体交换背后的分子事件的许多细节。而关于细胞中ER-高尔基体界面的组织仍然存在基本问题。蝇脂肪体及翅膀盘的内质网-高尔基体结构组成，发现了参与双向运输的、与高尔基体相连的前顺式高尔基体区域，在内质网出口-高尔基体区域内发现大量的囊泡和珠状小管，且没有发现巨型运输载体。本研究提示果蝇同时存在囊泡运输和管状运输，为从进化角度进一步理解蛋白质早期分泌途径以及胶原等巨型货物的运输机制提供了重要依据。.在跨高尔基体，除了主要的高尔基体到质膜分泌途径外，还存在与内溶酶体系统的复杂交通连接。我们研究了显示自噬囊泡中分泌货物积累的ESCRT-III组分Vps20, SNARE结合Rop和溶酶体泵亚基VhaPPA1-1。发现Vps20, Rop和溶酶体标记位于反式高尔基体附近(是溶酶体的主要细胞位置)，并且早期、晚期和回收内体也与反式高尔基体相关的降解区室相关联，其中发生分泌货物和其他材料的基底微自噬。这个隔间的破坏会导致我们命中的货物堆积，包括Munc18同系物Rop, Syx1和Syx4需要Rab11介导的内体回收。除了基础微自噬外，还表明反式高尔基体相关的降解区室有助于发育和饥饿诱导的巨自噬中自噬囊泡的生长。果蝇反式高尔基体是整个内膜系统的引力中心。.果蝇背侧和腹侧翅膀表面在一个矛盾的过程中贴合、分离、再贴合，涉及到细胞外基质的粘附、基质的产生和降解以及基质-细胞骨架间的协作。在果蝇翅膀中开展了针对细胞骨架相关蛋白的遗传筛选实验。我们发现Shot、Patronin、septins和在翅膀特异表达的新蛋白Sdb(SAXO downstream of blistered)对于形成能够有效抗压的细胞骨架突起连接是必需的。采用最新的果蝇遗传和转基因技术还发现了Sdb是受到翅脉间转录因子SRF(Serum Response Factor)调控的微管稳定蛋白。能够产生抗压的电缆般细胞骨架是有翅昆虫在进化上的关键一步。.损伤诱导的免疫反应的主要介质是JAK/STAT信号传导。果蝇Upd细胞因子的JAK/STAT激活在多器官的发育中多效性地参与。JAK/STAT的炎症和发育作用是否相交尚不清楚。我们发现JAK/STAT在控制的前胸腺PG中起作用。组织损伤和肿瘤也激活了PG中的JAK/STAT并延迟蜕变。