The role of RGP1 in extracellular matrix secretion and chondrocyte cell shape development

细胞产生并分泌蛋白质到细胞外空间，这些蛋白质组装形成细胞外基质。 矩阵（ECM）。ECM对于生长、伤口愈合和结构发育至关重要。缺陷 ECM蛋白本身或其相关的分泌机制经常表现在临床表型中。 许多分泌机制是在酵母中发现的。作为单细胞生物，酵母不分泌 ECM蛋白。因此，分泌机制在ECM分泌中的作用在很大程度上仍未被探索。 在酵母和哺乳动物细胞中，一种被称为Rgp 1的蛋白质被证明可以结合Ric 1，并作为一种蛋白质发挥作用。 Rab 6的鸟嘌呤核苷酸交换因子（GEF）复合物。然而，Rgp 1的作用还没有得到证实。 在多细胞生物体中原位检测，并且没有脊椎动物模型可用于研究其功能。 为了了解Rgp 1在脊椎动物中的作用，我使用CRISPR/Cas9基因组编辑来生成稳定的Rgp 1基因。 rgp 1-null（rgp 1-/-）斑马鱼品系。 我的初步研究表明，在缺乏Rgp 1的情况下，斑马鱼胚胎表现出 畸形颅面软骨，主要由软骨细胞组成的组织。rgp 1缺陷型 软骨细胞在细胞内积累胶原蛋白，并采用双凹盘状形态，表明软骨细胞是一种 Rgp 1在多细胞生物中的功能更为复杂。斑马鱼和人类RGP 1的序列 序列是高度保守的，因此，我预测在斑马鱼中的发现将为Rgp 1生物学提供信息 在人类和其他脊椎动物中。 该项目的目的是评估Rgp 1在ECM分泌中的作用，并确定Rgp 1在ECM分泌中的作用。 Rgp 1在软骨细胞形态发育中的作用。这个项目使用基因操作， 免疫荧光和活细胞成像实验，以确定Rgp 1的亚细胞功能， 更好地理解似乎不相连的贩运和细胞形状途径是如何通过 单一蛋白质这项研究将有助于解释蛋白质运输，ECM沉积，细胞 形状变化和颅面骨骼发育。

Cells produce and secrete proteins to the extracellular space that assemble to form an extracellular matrix (ECM). The ECM is essential for growth, wound healing, and structural development. Defects in the ECM proteins themselves or their associated secretory machinery often manifest in clinical phenotypes. Much of the secretory machinery was discovered in yeast. Yeast, as single-cell organisms, do not secrete ECM proteins. Thus, the role of secretory machinery in ECM secretion has remained largely unexplored. A protein known as Rgp1 had been shown in yeast and mammalian cells to bind Ric1 and function as a guanine nucleotide exchange factor (GEF) complex for Rab6. However, the role of Rgp1 has not been examined in situ in multicellular organisms, and no vertebrate model was available to study its function. To understand the role of Rgp1 in a vertebrate, I used CRISPR/Cas9 genome editing to generate a stable rgp1-null (rgp1-/-) zebrafish line. My preliminary studies have shown that in the absence of Rgp1, zebrafish embryos manifest with malformed craniofacial cartilage, a tissue primarily composed of chondrocytes. The rgp1-deficient chondrocytes accumulate collagen intracellularly and adopt a biconcave discoid morphology, suggesting a more complex function of Rgp1 in multicellular organisms. The sequences of zebrafish and human RGP1 sequences are highly conserved; therefore, I predict that findings in zebrafish will inform on Rgp1 biology in humans and other vertebrates. The aims of this project are to assess the role of Rgp1 in ECM secretion and to determine the role of Rgp1 in chondrocyte cell shape development. This project uses genetic manipulations, immunofluorescence, and live cell imaging experiments to determine subcellular functions of Rgp1 to better understand how seemingly disconnected trafficking and cell shape pathways are regulated through a single protein. This study will help explain a connection between protein trafficking, ECM deposition, cell shape changes, and craniofacial skeleton development.