Epithelial cells contain noncentrosomal microtubules (MTs), whose minus ends are oriented apically. In contrast with the well-known interactions of the minus ends with the centrosome, little is known about the termination site of the noncentrosomal minus ends. Here we show that a population of MT minus ends is anchored at the zonula adherens (ZA), the apical-most part of the cadherin-based adherens junction, via a protein that we have termed Nezha. We initially identified PLEKHA7 as a ZA component and subsequently detected Nezha as a partner for PLEKHA7. Nezha bound MTs at their minus ends and tethered them to the ZA. Furthermore, we found that a minus end-directed motor, KIFC3, was concentrated at the ZA in a PLEKHA7/Nezha/MT-dependent manner; and depletion of any of these proteins resulted in disorganization of the ZA. We propose that the PLEKHA7/Nezha/MT complex regulates the ZA integrity by recruiting KIFC3 to this junctional site.

No abstract

Major microtubules in epithelial cells are not anchored to the centrosome, in contrast to the centrosomal radiation of microtubules in other cell types. It remains to be discovered how these epithelial microtubules are generated and stabilized at noncentrosomal sites. Here, we found that Nezha [also known as calmodulin-regulated spectrin-associated protein 3 (CAMSAP3)] and its related protein, CAMSAP2, cooperate in organization of noncentrosomal microtubules. These two CAMSAP molecules coclustered at the minus ends of noncentrosomal microtubules and thereby stabilized them. Depletion of CAMSAPs caused a marked reduction of microtubules with polymerizing plus ends, concomitantly inducing the growth of microtubules from the centrosome. In CAMSAP-depleted cells, early endosomes and the Golgi apparatus exhibited irregular distributions. These effects of CAMSAP depletion were maximized when both CAMSAPs were removed. These findings suggest that CAMSAP2 and -3 work together to maintain noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome, and that the network of CAMSAP-anchored microtubules is important for proper organelle assembly.

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.