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.
Highlights
Drosophila homologue of Diaphanous (Diaph)-proteins belong to the family of formins, which stimulate both actin nucleation and microtubule (MT) stabilization[1,2,3]
In the absence of extracellular stimuli, the actin nucleating activity of Diaph formins is auto-inhibited through interaction of the C-terminal Diaphanous Autoregulatory Domain (DAD) with the N-terminal Diaphanous Inhibitory Domain (DID)
We addressed the functional role of human Diaph[2] for progression of colorectal carcinoma cells and its potential function in chromosome alignment, metaphase progression and MT-modification
Summary
Drosophila homologue of Diaphanous (Diaph)-proteins belong to the family of formins, which stimulate both actin nucleation and microtubule (MT) stabilization[1,2,3]. A related study confirmed the essential role for Diaph[2] for chromosome alignment and revealed that the isolated FH1/FH2-domains of Diaph[2] stabilize MTs in an Aurora-B dependent manner[6]. From these data the authors concluded that Diaph[2] controls chromosome alignment by stabilizing spindle MTs. Since failures in chromosome alignment can result in unequal distribution of chromosomes and in chromosomal instability (CIN)[13], it seems crucial to investigate if Diaph[2] is involved in the control of CIN in colon cancer. We addressed the functional role of human Diaph[2] for progression of colorectal carcinoma cells and its potential function in chromosome alignment, metaphase progression and MT-modification
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