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

细胞产生和分泌蛋白质到细胞外空间，然后组装形成细胞外 矩阵(ECM)。细胞外基质对生长、伤口愈合和结构发育是必不可少的。产品中的缺陷 细胞外基质蛋白本身或与其相关的分泌机制经常出现在临床表型中。 许多分泌机制都是在酵母中发现的。酵母，作为单细胞有机体，不分泌 ECM蛋白。因此，分泌机制在细胞外基质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发现了一种名为Rgp1的蛋白质，它能与RIC1结合并发挥RIC1的功能 鸟嘌呤核苷酸交换因子与Rab6的复合体。然而，Rgp1的作用并不是 在多细胞生物体中进行了原位检测，没有脊椎动物模型可以用来研究它的功能。 为了了解Rgp1在脊椎动物中的作用，我使用CRISPR/Cas9基因组编辑来产生稳定的 Rgp1-空(rgp1-/-)斑马鱼线。 我的初步研究表明，在缺乏Rgp1的情况下，斑马鱼胚胎表现出 畸形的头面部软骨，主要由软骨细胞组成的组织。Rgp1基因缺失 软骨细胞在细胞内积聚胶原蛋白，呈双凹盘状形态，提示 Rgp1在多细胞生物中的更复杂的功能。斑马鱼和人类RGP1的序列 序列高度保守；因此，我预测在斑马鱼中的发现将为Rgp1生物学提供信息 在人类和其他脊椎动物身上。 该项目的目的是评估Rgp1在ECM分泌中的作用，并确定其作用 Rgp1在软骨细胞形态发育中的作用。这个项目使用了基因操作， 免疫荧光和活细胞成像实验确定Rgp1的亚细胞功能 更好地理解看似分离的贩运和细胞形状通路是如何通过 一种单一的蛋白质。这项研究将有助于解释蛋白质运输、细胞外基质沉积、细胞 形状变化，头面部骨骼发育。

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.