In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our
in vitro
results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.
Detyrosination is a major post-translational modification of microtubules (MTs), which has significant impact on MT function in cell division, differentiation, growth, migration, and intracellular trafficking. Detyrosination of α-tubulin occurs mostly via the recently identified complex of vasohibin1/2 (VASH1/2) and small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of VASH1/2:SVBP, and little is known about the regulation of detyrosination. Here, we found that intracellular calcium is required for efficient MT detyrosination. Furthermore, we show that calcium-dependent proteases calpains 1 and 2 regulate MT detyrosination in VASH1:SVBP overexpressing human embryonal kidney (HEK293T) cells. We identified new calpain cleavage sites in the N-terminal disordered region of VASH1. However, this cleavage did not affect the enzymatic activity of VASH. In conclusion, we suggest that the regulation of VASH1-mediated MT detyrosination by calpains could occur independent of VASH catalytic activity or via another yet unknown tubulin carboxypeptidase. Importantly, calpains’ calcium dependency could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for basic and clinical research.
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