Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

Abstract

SummaryThe role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr-and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.

Kinesin-2 mediates apical endosome transport during epithelial lumen formation

“…To test this possibility, we first analyzed FIP5-GFP distribution in Matrigel-embedded cells treated with 10 μM of nocodazole for 45 min. It has been previously shown that nocodazole treatment causes depolymerization of dynamic microtubules, while having no effect on acetylated microtubules, such as the central spindle 15 , 16 . Consistent with the previous studies using filter-grown MDCK cells, 8 nocodazole treatment in interphase cells caused dispersal of FIP5-containing apical endosomes, while maintaining their enrichment at the apical pole (Fig.…”

Post-translational modifications of tubulin: pathways to functional diversity of microtubules

“…For example, detyrosination affects MT interactions with intermediate filaments [24] and microfilaments in vivo [18]. In addition, detyrosination is associated with differentiation and polarization of some cells [25, 26] (Fig. 3).…”

“…3). In these studies, subconfluent cells have moderate levels of detyrosination, which increases with confluency and declines as confluent cells become polarized [25, 26]. Local reversibility of detyrosination generates tubulin and MTs with distinct properties more efficiently than protein synthesis and degradation.…”

Dissecting the role of the tubulin code in mitosis

“…However, the discrimination between kMTs and ipMTs is difficult and their precise tubulin composition at each stage of mitosis remains unclear. Moreover, immunofluorescence analysis to detect detyrosinated α-tubulin has revealed continuous and discontinuous regions along the MT length, suggesting the existence of alternate stretches of detyrosinated and tyrosinated tubulin, whose functional meaning remains unknown (Geuens et al, 1986; Zink et al, 2012). …”