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.

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

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.

Multicellular rosette formation during cell ingression in the avian primitive streak.

Cell movements are a fundamental feature during the development of multi-cellular organisms. In amniote gastrulation, cells ingress through the primitive streak, which identifies the anterior-posterior axis of the embryo. We investigated the cytoskeletal architecture during these morphogenetic processes and characterized microtubule organisation in whole chick embryos. This revealed the distribution of cells with polarized and radial microtubule (MT) arrays across different regions of the embryo. Cells in the epiblast usually displayed radial MT-arrays, while the majority of cells in the primitive streak had polarized MT-arrays. Within the primitive streak, many cells organized into groups and were arranged in rosette-like structures with a distinct centre characterized by an accumulation of actin. Extended confocal microscopy and three-dimensional image reconstruction identified tips of polarized cells that were protruding from the plane of rosettes, usually from the centre. We propose that organization into higher order structures facilitates cell ingression during gastrulation.

Centrosomal CAP350 protein stabilises microtubules associated with the Golgi complex.

A comprehensive model of how the centrosome organises the microtubule network in animal cells has not yet been elucidated. Here we show that the centrosomal large CAP-Gly protein CAP350 is not only present at the centrosome, but is also present as numerous dots in the pericentrosomal area. Using in vitro and in vivo expression of partial constructs, we demonstrated that CAP350 binds microtubules through an N-terminal basic region rather than through its CAP-Gly domain. CAP-Gly-containing domains of CAP350 are targeted not only to the centrosome but also to a Golgi-like network. Interestingly, full-length GFP-tagged CAP350 bound preferentially to microtubules in the pericentrosomal area. These results indicate that the large CAP350 protein has a dual binding ability. Overexpression of CAP350 promoted an increase in the stability of the whole microtubule network, as judged by a significant decrease in the number of EB1 comets and by an enhanced microtubule resistance to Nocodazole treatment. In support of this, CAP350 depletion decreased microtubule stability. Moreover, both depletion and overexpression of CAP350 induced specific fragmentation of the Golgi complex while maintaining a juxtanuclear localisation. We propose that CAP350 specifically stabilises Golgi-associated microtubules and in this way participates in the maintenance of a continuous pericentrosomal Golgi ribbon.

Ninein is released from the centrosome and moves bi-directionally along microtubules.

Cell-to-cell contact and polarisation of epithelial cells involve a major reorganisation of the microtubules and centrosomal components. The radial microtubule organisation is lost and an apico-basal array develops that is no longer anchored at the centrosome. This involves not only the relocation of microtubules but also of centrosomal anchoring proteins to apical non-centrosomal sites. The relocation of microtubule minus-end-anchoring proteins such as ninein to the apical sites is likely to be essential for the assembly and stabilisation of the apico-basal arrays in polarised epithelial cells. In this study, we establish that ninein is highly dynamic and that, in epithelial cells, it is present not only at the centrosome but also in the cytoplasm as distinct speckles. Live-cell imaging reveals that GFP-ninein speckles are released from the centrosome and move in a microtubule-dependent manner within the cytoplasm and thus establishes that epithelial cells possess the mechanical means for relocation of ninein to non-centrosomal anchoring sites. We also provide evidence for the deployment of ninein speckles to apical anchoring sites during epithelial differentiation in both an in situ tissue and an in vitro culture system. In addition, the findings suggest that the non-centrosomal microtubule anchoring sites associate with adherens junctions in polarised epithelial cells.