Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells

Abstract

Apico-basal polarisation of epithelial cells involves a dramatic reorganisation of the microtubule cytoskeleton. The classic radial array of microtubules focused on a centrally located centrosome typical of many animal cells is lost or greatly reduced and a non-centrosomal apico-basal array develops. The molecules and mechanisms responsible for the assembly and positioning of these non-centrosomal microtubules have not been fully elucidated. Using a Nocodazole induced regrowth assay in invitro culture (MDCK) and in situ epithelial (cochlear Kolliker's) cell models we establish that the apico-basal array originates from the centrosome and that the non-centrosomal microtubule minus-end anchoring sites do not contribute significantly to their nucleation. Confocal and electron microscopy revealed that an extended radial array assembles with microtubule plus-ends targeting cadheren sites at adherens junctions and EB1 and CLIP-170 co-localising with beta-catenin and dynein clusters at the junction sites. The extended radial array is likely to be a vital intermediate step in the assembly process with cortical anchored dynein providing the mechanical force required for microtubule release, translocation and capture. Ultrastructural analyses of the apico-basal arrays in fully polarised MDCK and Kolliker's cells revealed microtubule minus-end association with the most apical adherens junction (Zonula adherens). We propose that a release and capture model involving both microtubule plus- and minus-end capture at adherens junctions is responsible for the generation of non-centrosomal apico-basal arrays in most centrosome containing polarised epithelial cells.