Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.阿法丁蛋白（afadin）是一种肌动蛋白丝结合蛋白，可与细胞黏附分子nectin（Nectin）协同介导细胞黏附过程，还可通过不依赖nectin的途径，与小G蛋白Rap1协同调控细胞定向迁移。本研究采用阿法丁蛋白条件性基因敲除（conditional gene knockout，cKO）小鼠，探究了阿法丁蛋白在小肠上皮组织构建中的功能。阿法丁蛋白定位于整个隐窝-绒毛轴（crypt-villus axis）中各类上皮细胞的黏着连接（adherens junctions）处。对照组小鼠的潘氏细胞（Paneth cells）定位于隐窝基底且局限于此区域，但在阿法丁蛋白条件性基因敲除小鼠中，潘氏细胞未被限制在该区域，反而迁移至绒毛内部。该突变小鼠体内其他类型上皮细胞的分布无显著变化。残留在隐窝内的潘氏细胞形态异常，被掩埋于相邻细胞之间，且不朝向肠腔。此类细胞的黏着连接与紧密连接（tight junctions）的形成受到损伤。Rap1与EphB3在对照组潘氏细胞中呈高表达状态，但在阿法丁蛋白缺陷的潘氏细胞中表达显著下调。综上，本研究结果表明，阿法丁蛋白可通过维持潘氏细胞与相邻隐窝细胞的黏附，并抑制其向绒毛顶端迁移，从而维持潘氏细胞在隐窝基底的限制性定位。