Abstract
Epithelial monolayer formation depends on the architecture and composition of the microtubule cytoskeleton. Microtubules control bidirectional trafficking and determine the positioning of structural cellular proteins. We studied the role of tubulin tyrosination in epithelial cell shape and motility. Tubulin tyrosine ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, was depleted or overexpressed in 2D epithelial monolayers as well as in 3D intestinal organoids. We demonstrate qualitatively and quantitatively that in the absence of TTL the cells comprise high levels of detyrosinated tubulin, change their shape into an initial flat morphology and retardedly acquire a differentiated columnar epithelial cell shape. Enhanced adhesion and accelerated migration patterns of TTL-knockout cells combined with reverse effects in TTL-overexpressing cells indicate that the loss of TTL affects the organization of cell adhesion foci. Precipitation of detyrosinated tubulin with focal adhesion scaffold components coincides with increased quantities and persistence of focal adhesion plaques. Our results indicate that the equilibrium between microtubules enriched in detyrosinated or tyrosinated tubulin modulates epithelial tissue formation, cell morphology, and adhesion.
Highlights
Dynamic arrangement and structured architecture of cytoskeletal elements ensure formation and maintenance of epithelial cell sheets
Line scan analysis of α-tubulin intensities revealed that a central, perinuclear packaging of microtubules, which was very prominent in Madin-Darby Canine Kidney (MDCK) tubulin tyrosine ligase (TTL) cells, was shifted to a peripheral accumulation in MDCKTTL−GFP cells (Figure 1C and Supplementary Figure 1A)
MDCK TTL cells had an outspread and flat morphology and slowly reached a height of only about 4 μm after 5 days in culture, which was rescued by TTL-GFP-expression (Figure 1D)
Summary
Dynamic arrangement and structured architecture of cytoskeletal elements ensure formation and maintenance of epithelial cell sheets. Microtubules can be modulated by posttranslational modifications, which include acetylation, tyrosination, detyrosination, 2 modification, polyglutamylation, palmitoylation and phosphorylation (Janke and Magiera, 2020; Roll-Mecak, 2020). Tyrosinated (tyr-tubulin), and detyrosinated (detyr-tubulin) α-tubulin is generated by a cycle of removal and subsequent religation of tyrosine to the carboxy terminus of this polypeptide. Detyrosination can inhibit microtubule disassembly, whereas dynamic microtubules are predominantly tyrosinated (Palazzo et al, 2004). The vasohibins VASH1 and its homolog VASH2 have been identified to remove the C-terminal tyrosine of α-tubulin (Aillaud et al, 2017; Nieuwenhuis et al, 2017). The tubulin tyrosination cycle is completed by the enzyme tubulin tyrosine ligase (TTL), which catalyzes C-terminal α-tubulin tyrosination on αβtubulin heterodimers and restores tyr-tubulin (Raybin and Flavin, 1975).
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