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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.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel best known for its ability to be gated by the pungent constituent of red chili pepper, capsaicin, and related chemicals from the group of vanilloids as well as by noxious heat. As such, it is mostly expressed in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Its activation is also sensitized by the numerous endogenous inflammatory mediators and second messengers, making it an important determinant of nociceptive signaling. Except for such signaling, though, neuronal TRPV1 activation may influence various organ functions by promoting the release of bioactive neuropeptides from sensory fiber innervation organs. However, TRPV1 is also found outside the sensory nervous system in which its activation and function is not that straightforward. Thus, TRPV1 expression is detected in skeletal muscle; in some types of smooth muscle; in epithelial and immune cells; and in adipocytes, where it can be activated by the combination of dietary vanilloids, endovanilloids, and pro-inflammatory factors while the intracellular calcium signaling that this initiates can regulate processes as diverse as muscle constriction, cell differentiation, and carcinogenesis. The purpose of the present review is to provide a clear-cut distinction between neurogenic TRPV1 effects in various tissues consequent to its activation in sensory nerve endings and non-neurogenic TRPV1 effects due to its expression in cell types other than sensory neurons.

Little is known about how mitotic cells round against epithelial confinement. At the beginning of mitosis, cells markedly change their morphology as they round up1. During mitotic cell rounding, the microtubule cytoskeleton forms the mitotic spindle, a central machinery that captures and organizes chromosomes2. Mitotic cell rounding occurs in the vast majority of animal cells3 and plays a role in maintaining tissue organization. Here, we have engineered micropillar arrays with spatial and mechanical properties to mimic the mechanical confinement of epithelia and to measure the forces generated by single epithelial cells rounding for mitosis. For our studies we use Madin?Darby canine kidney (MDCK) cell lines that are commonly used as a model for epithelial cells. We observe that MDCK cells deflect nearby micropillars as they round and thereby create sufficient space for mitosis.However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells4,5. The availability of large pillar arrays and the optical readout of the assay enables the simultaneous characterization of hundreds of single cells to investigate the cellular mechanisms that drive the drastic shape change in mitosis.We have demonstrated that the contributions of proteins involved in mitotic cell rounding to generating force can be quantified by combining the micropillar assay with targeted chemical perturbation. Thus, the micropillar assay opens up the possibility to perform a large-scale screen for genes and mechanisms involved in mitotic cell rounding.References:1. McConnell, C. H. Mitosis in hydra - mitosis in the ectodermal epitheliomuscular cells of Hydra. Biol. Bull. 64, 86?95 (1933).2. Cramer, L. P. & Mitchison, T. J. Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding. Mol. Biol. Cell 8, 109?119 (1997).3. Kline-Smith, S. L. & Walczak, C. E. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol. Cell 15, 317?327 (2004).4. Stewart, M. P. et al. Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226?230 (2011).5. Sorce, B. et al. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement. Nature Commun. 25, 8872-8876 (2015)