Tubb4b is required for multi-ciliogenesis in the mouse.

Cilia are microtubule (MT)-based organelles present on the surface of nearly all vertebrate cells. MTs are polymers of α- and ß-tubulins that are each encoded by multiple, individual isotype genes. Tubulin isotype composition is thought to influence MT behaviors. Ciliary MTs differ from other MTs in the cell in terms of organization, stability and post-translational modifications. However, little is known about the tubulin isotypes that build ciliary MTs and the functional requirements for tubulin isotypes in cilia have not been examined in vertebrates. Here, we have tested the role of the ß-tubulin isotype genes in the mouse that harbor a conserved amino acid motif associated with ciliated organisms. We found that Tubb4b localizes to cilia in multi-ciliated cells (MCCs) specifically. In respiratory and oviduct MCCs, Tubb4b is asymmetrically localized within multi-cilia, indicating that the tubulin isotype composition changes along the length of the ciliary axonemal MTs. Deletion of Tubb4b resulted in striking structural defects within the axonemes of multi-cilia, without affecting primary cilia. These studies show that Tubb4b is essential for the formation of a specific MT-based subcellular organelle and sheds light on the requirements of tubulin isotypes in cilia.

The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling.

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting forces generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation. Canoe's mammalian homologue Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We take these insights back to the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found that Canoe helps cells maintain junctional-cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation, and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe, many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homologue Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest that Canoe and Polychaetoid stabilize Bazooka/Par3 at cell-cell junctions, helping maintain balanced apical contractility and tissue integrity.