The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain

Abstract

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.

A cancer stem cell-like phenotype is associated with miR-10b expression in aggressive squamous cell carcinomas

“…5a), together with the fact that it was listed as a putative target of miR-10b. Its potential disease-relevance was substantiated by its recent association with colon carcinoma, and its potential role in actin-organization and microtubule stabilization [35][36][37]. When re-analyzing normalized RNA-seq data generated by Cho et al, retrieved from GEOrepository (GSE111582), both, DIAPH2 and HOXD10, showed lower expression in RDEB-cSCC versus RDEB-skin tissue [8], ( Supplementary Fig.…”

MiRNA-10b marks aggressive squamous cell carcinomas, and confers a cancer stem cell-like phenotype

“…We nominated diaphanous related formin 2 ( DIAPH2) for further evaluation based on significant differences in Kaplan Meier survival curves (p < 0.05, log-rank test, Figure 5A), together with the fact that it was listed as a putative target of miR-10b. Its potential disease-relevance was substantiated by its recent association with colon carcinoma, and its potential role in actin-organization and microtubule stabilization (35-37). When re-analyzing normalized RNA-seq data generated by Cho et al , retrieved from GEO-repository (GSE111582), both, DIAPH2 and HOXD10 , showed lower expression in RDEB-SCC versus RDEB-skin tissue (8, Supplementary Fig.…”

Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells

“…Although formin family proteins are known as actin polymerization promoters highly conserved in eukaryotes, its microtubule-related functions have also been reported [ 22 , 23 , 24 , 25 , 26 ]. For example, Daam and Inf2 are involved in microtubule acetylation, and Diaph2 , an isoform of Diaph3 , is involved in microtubule dynamics [ 25 , 27 ]. To test whether Diaph3 also has microtubule-related functions, we focused on acetylated α-tubulin, a sign of stabilized microtubules, and observed its level.…”

“…In addition, based on the results of multinucleation analysis using the mutant DIAPH3 ( Figure 5 B), it is likely that I704 and I733 are involved in the destabilization of the microtubule. On the other hand, DIAPH2 has been reported to be involved in microtubule dynamics without the FH2 domain [ 27 ], and it is possible that DIAPH3 possesses functional domains for microtubule regulation other than the FH2 domain. The analysis of DIAPH3 domains by using deletion mutants will help elucidate the function of Diaph3 in cytokinesis.…”