Abstract:
Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.
摘要:
柱状上皮细胞的分化涉及微管(MT)和中心体成分的急剧重组,形成不再锚定在中心体的顶端基底阵列。相反,MT的负端锚定在顶端非中心体微管组织中心(n-MTOC)。n-MTOC的形成至关重要,因为它们决定了MT的空间组织,进而影响细胞的形状和功能。然而,人们对它们是如何形成的知之甚少。我们之前已经表明,中心体锚定蛋白ninein从中心体释放,在上皮分化过程中以微管依赖性方式移动并在n-MTOC处积累。这里,我们报告使用耗竭和敲除(KO)方法,ninein的表达对于尖端基底阵列形成和上皮伸长至关重要,并且CLIP-170对于其重新部署到n-MTOC是必需的。功能抑制还显示IQGAP1和活性Rac1与CLIP-170协调,以促进微管加端皮质靶向和ninein重新部署。来自Clip1/Clip2双KO小鼠的肠组织和体外类器官,分别在编码CLIP-170和CLIP-115的基因中缺失,确认了CLIP-170对n-MTOC招募ninein的要求,尽管ninein在n-MTOC中丢失,但可能通过其他锚定因素(例如p150Glued和CAMSAP2)进行补偿,以确保顶端基底微管的形成。