探究TAPT1参与原生纤毛生成的分子机制及对骨发育的影响

进化保守的TAPT1蛋白，其同源蛋白在拟南芥中称为POD1，可作为参与内质网质量控制的分子伴侣指导拟南芥中花粉管导向。人TAPT1的突变导致骨软骨发育不良。我们发现，TAPT1定位于纤毛及其过渡区；TAPT1与膜相关，且与过渡区蛋白TCTN3相互作用；敲除TAPT1影响原生纤毛生成，并且在纤毛囊泡内出现小囊泡的积累。基于上述发现，本课题将利用敲除TAPT1的小鼠胚胎成纤维细胞和人视网膜上皮细胞，阐明TAPT1是否是一种新的膜栓系因子，研究TAPT1影响原生纤毛生成的分子机制；利用Osx1-Cre介导的成骨细胞特异性Tapt1基因敲除小鼠模型，在细胞和整体水平探究TAPT1与骨细胞纤毛形成及骨发育的关系。本项目的完成将为阐明纤毛过渡区蛋白参与原生纤毛生成并影响骨发育的分子机制提供理论依据，同时有利于对理解与膜相关的纤毛蛋白在不同物种间的功能相似性提供科学思路。

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein (TAPT1, also named POD1 in Arabidopsis thaliana) has been reported to be associated with endoplasmic reticulum (ER) membrane via its interaction with CALRETICULIN3 (CRT3), and acts as a chaperone involved in ER quality control for pollen tube guidance in Arabidopsis, and its mutations in human result in osteochondrodysplasia. .We have found that TAPT1, required for primary cilium formation, localized to the ciliary transition zone and along the cilia. TAPT1 binds to the transition zone protein TCTN3. Transmission electron microscopy (TEM) displayed several vesicles inside a ciliary vesicle (CV) of a TAPT1-null cell. Subcellular fractionation of HeLa cells revealed that TAPT1, behaving as peripheral membrane protein, was mainly associated with membranes, from which they could be extracted by buffers containing a high salt concentration. .The aforementioned findings promote us to address the following questions. (1) Is the distribution of proteins localizing to the transition zone and cilia affected in Tapt1-knockout MEFs and TAPT1-depleted RPE-1 cells? (2) Is TAPT1 a novel membrane-tethering factor responsible for the ciliary receptor-containing vesicles tethered to the ciliary transition zone? (3) Does Osx1-Cre-mediated conditional deletion of Tapt1 in the osteoblast lineage result in defective cilium formation in mouse tibia bone? (4) Does the defective ciliogenesis contribute to skeletal abnormalities in osteoblast-specific Tapt1 knockout mice?.Results from the research will provide key mechanistic insights to explicate function of a transition zone protein in primary cilium formation and its involvement in ciliopathy, and enhance our understanding of ciliary proteins functionally conserved across species.