CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells

Abstract

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.

Control of apico–basal epithelial polarity by the microtubule minus-end-binding protein CAMSAP3 and spectraplakin ACF7

“…1A). In agreement with previous publications (Meng et al, 2008;Tanaka et al, 2012;Toya et al, 2016), in this cell line, CAMSAP3-GFP localized to microtubule ends, colocalized with endogenous CAMSAP2 and was concentrated at the apical side of polarized Caco-2 monolayers ( Fig. 1B-D).…”

“…Intestinal epithelial cells of those mice could still polarize and form a normal brush border, in spite of the fact that the microtubules were disorganized, and the nuclei and Golgi membranes mispositioned (Toya et al, 2016). Experiments in Caco-2 cells, a human intestinal cancer cell line, have demonstrated that the first coiled-coil region of CAMSAP3 is involved in recruitment of CAMSAP3-bound microtubule minus ends to the apical side (Toya et al, 2016). In CAMSAP1, this coiled-coil region was shown to bind to spectrin (King et al, 2014).…”

“…Through the CKK domain and the adjacent positively charged microtubule-binding regions, CAMSAP3, as well as its homolog CAMSAP2 recognize and decorate polymerizing microtubule minus ends, forming stretches of stabilized microtubule lattice that can serve as 'seeds' for non-centrosomal microtubule outgrowth (Jiang et al, 2014). Recent work shows that CAMSAP3 is an important factor in organizing apico-basal microtubule arrays in enterocytes, where it strongly localizes to the apical cell cortex and tethers microtubule minus ends (Toya et al, 2016). Mice homozygous for the Cterminal truncation of CAMSAP3, in which the CKK domain is lost, are viable but show increased lethality at early postnatal stages (Toya et al, 2016).…”

“…Recent work shows that CAMSAP3 is an important factor in organizing apico-basal microtubule arrays in enterocytes, where it strongly localizes to the apical cell cortex and tethers microtubule minus ends (Toya et al, 2016). Mice homozygous for the Cterminal truncation of CAMSAP3, in which the CKK domain is lost, are viable but show increased lethality at early postnatal stages (Toya et al, 2016). Intestinal epithelial cells of those mice could still polarize and form a normal brush border, in spite of the fact that the microtubules were disorganized, and the nuclei and Golgi membranes mispositioned (Toya et al, 2016).…”

“…Mice homozygous for the Cterminal truncation of CAMSAP3, in which the CKK domain is lost, are viable but show increased lethality at early postnatal stages (Toya et al, 2016). Intestinal epithelial cells of those mice could still polarize and form a normal brush border, in spite of the fact that the microtubules were disorganized, and the nuclei and Golgi membranes mispositioned (Toya et al, 2016). Experiments in Caco-2 cells, a human intestinal cancer cell line, have demonstrated that the first coiled-coil region of CAMSAP3 is involved in recruitment of CAMSAP3-bound microtubule minus ends to the apical side (Toya et al, 2016).…”