Regulatory mechanisms that control vesicle secretion at the endoplasmic reticulum

摘要 目前资助的项目的长期目标是定义 调节早期分泌途径中细胞器的空间分布并确定 这种结构对细胞生长和正常膜运输的重要性 发展。大多数用于分泌的生物合成货物分子开始了他们的旅程 在内质网(ER)的特定亚域内，称为内质网出口部位。在这些地方 地点，首先生成COPII涂层载体，包装货物以运输到ER- 高尔基体中间隔(ERGIC)，与内质网出口并列的稳定细胞器 网站。COPII外壳由两个多聚体蛋白质复合体Sec23/24和Sec23/24组成 Sec13/31和小的GTP酶Sar1。尽管这些因素足以重建 在体外，囊泡从化学定义的膜中萌发，需要额外的蛋白质来 促进COPII囊泡的生物生成和细胞内的顺行转运。我们目前获得的资金 该项目的重点是TFG的作用，TFG是一种后生动物特有的蛋白质，是正常 贩运涂有COPII的运输船。根据我们的初步结果，我们假设 TFG在ER/ERGIC接口形成高度受监管的网络，从而促进本地 保留内质网衍生的小泡，为COPII涂层的分解和 随后与ERGIC膜融合。重要的是，TFG的突变一直是 与进行性神经退行性疾病有关，包括遗传性痉挛截瘫 (HSP)，提示COPII介导的转运在维持神经元功能方面发挥了作用。这 修订申请旨在扩大我们项目的范围。具体地说，我们的目标是 设计新的过敏性紫杉醇大鼠模型并确定关键细胞器突变的机制 成分包括TFG(SPG57)、atlastin-1(SPG3A)和ESCRT-I亚单位Vps37A (SPG53)，导致皮质脊髓束轴索病变。这些研究充分利用了我们之前的 在大鼠中成功地使用CRISPR介导的基因组编辑并试图定义两者共享 以及独特的病理特征，观察到的是影响ER和 内体动态平衡，这是已知的神经退行性疾病的基础。

Abstract The long-term goals of the currently funded project are to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER), known as ER exit sites. At these locations, COPII-coated carriers are first generated, packaging cargoes for transport to ER- Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to ER exit sites. The COPII coat is composed of two multimeric protein complexes, Sec23/24 and Sec13/31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII vesicle biogenesis and anterograde transport in cells. Our currently funded project focuses on the role of TFG, a metazoan-specific protein required for the normal trafficking of COPII-coated transport carriers. Based on our preliminary results, we hypothesize that TFG forms a highly regulated meshwork at the ER/ERGIC interface that facilitates the local retention of ER-derived vesicles, providing sufficient time for COPII coat disassembly and subsequent fusion with ERGIC membranes. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, including hereditary spastic paraplegia (HSP), suggesting a role for COPII-mediated transport in maintaining neuron function. This revision application seeks to expand the scope of our project. Specifically, our goal is to engineer new rat models of HSP and define mechanisms by which mutations in key organelle components, including TFG (SPG57), atlastin-1 (SPG3A) and the ESCRT-I subunit Vps37A (SPG53), lead to axonopathy in the corticospinal tract. These studies leverage our previous successes in using CRISPR-mediated genome editing in rats and seeks to define both shared and unique pathological features observed as a result of mutations that impact ER and endosome homeostasis, which are known to underlie neurodegenerative disease in patients.