软骨细胞分化过程中大量胞外基质(ECM)的积累依赖于包括胶原蛋白在内的ECM蛋白的合成和转运，这些纤维状蛋白的长度超过300 nm，其在细胞内从内质网向高尔基体的转运需要装配专门的巨型囊泡。在软骨细胞分化中，这类负责转运大体积蛋白的巨型囊泡如何装配、如何选取和装填被运载的蛋白，是细胞生物学和发育生物学中关键但未解决的问题。在前期的工作中，我们发现SLY1（一个Sec domain蛋白）突变的斑马鱼胚胎由于软骨发生缺陷导致严重的头面部发育异常。进一步的分析表明SLY1突变的软骨细胞内，ECM蛋白无法从内质网向高尔基体转运，这些信息提示SLY1作为核心组份参与巨型囊泡的装配。我们计划利用斑马鱼胚胎和哺乳动物软骨细胞体外分化系统来探究在软骨细胞分化过程中，SLY1如何通过控制内质网－高尔基体中间囊泡与内质网之间的质膜融合来参与巨型囊泡的装配。

Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by up-regulation of effectors of protein transport to cope with both increased demand for transport of ECM components and the large size of molecules including procollagen. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we identified a mutant that disrupts the gene encoding Sec1 family domain containing 1 (sly1). Homozygous sly1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of sly1 was found to hinder ER to Golgi transport of ECM proteins. This preliminary data suggested SLY1 acts as central component in assembling of ECM-containing mega-carrier. In this proposal, we planned using zebrafish embryo and mammalian chondrocyte differentiation model to explore the molecular mechanisms by which sly1 involving in assembling of ECM-containing mega-carrier. We plan to address the following questions in our proposal. First, how SLY1 was recruited to ECM-enriched domain at the ER. Second, how SLY1 regulates the fusion between post-ER membrane compartment and ECM-enriched domain at the ER, which grows into an ECM-containing mega-carrier. We expect the results from this proposed study will provide evidence to the detail mechanism of ECM intracellular transferring and advance our understand for diseases such as osteogenesis imperfecta and congenital craniofacial abnormalities.