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Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

Microtubules (MTs) play essential roles in many steps of autophagy, an important degradation pathway in the maintenance of cellular homoeostasis. In many cells, MT networks are comprised of centrosomal MTs and noncentrosomal MTs. However, it is unknown whether noncentrosomal MTs and its binding proteins are involved in autophagy. Here, we show in HeLa cells that calmodulin-regulated spectrin-associated protein 2 (CAMSAP2), a noncentrosomal MT minus-end stabilizing protein, regulates retrograde transport of autophagosomes through MT dynamics. CAMSAP2 cooperates with EB1 to regulate end-binding protein 1 (EB1) behaviour at MT plus ends, MT growth directions and autophagosome transport. An association between CAMSAP2 and EB1 in the cytosol may modulate EB1 binding to MT plus ends. Collectively, our data indicate that noncentrosomal MTs regulate autophagy through a cross-talk between CAMSAP2 and EB1. [ABSTRACT FROM AUTHOR]

Summary For adaptation to complex cellular functions, dynamic cytoskeletal networks are required. There are two major components of the cytoskeleton, microtubules and actin filaments, which form an intricate network maintaining an exquisite cooperation to build the physical basis for their cellular function. However, little is known about the molecular mechanism underlying their synergism. Here, we show that in Caco2 epithelial cells, noncentrosomal microtubules crosstalk with F-actin through their minus ends and contribute to the regulation of focal adhesion size and cell migration. We demonstrate that ACF7, a member of the spectraplakin family of cytoskeletal crosslinking proteins, interacts with Nezha (also called CAMSAP3) at the minus ends of noncentrosomal microtubules and anchors them to actin filaments. Those noncentrosomal microtubules cooperate with actin filaments through retrograde flow to keep their length and orientation perpendicular to the cell edge as well as regulate focal adhesion size and cell migration. [ABSTRACT FROM AUTHOR]