Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres.

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.

Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour.

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin.

The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.