Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas.

Adhesion to extracellular matrix is required for cell cycle progression through the G1 phase and for the completion of cytokinesis in normal adherent cells. Cancer cells acquire the ability to proliferate anchorage-independently, a characteristic feature of malignantly transformed cells. However, the molecular mechanisms underlying this escape of the normal control mechanisms remain unclear. The current study aimed to identify adhesion-induced reactions regulating the cytokinesis of non-transformed human fibroblasts.The adhesion-dependent control of cytokinesis was found to occur at a late stage close to the abscission, during which the endosomal sorting complex required for transport (ESCRT) severs the thin intercellular bridge connecting two nascent daughter cells. CEP55, a key protein involved in the abscission process, was localized at the midbody in both adherent and non-adherent fibroblasts, but it was unable to efficiently recruit ALIX, TSG101, and consequently the ESCRT-III subunit CHMP4B was missing in the non-adherent cells. PLK1, a kinase that prevents premature recruitment of CEP55 to the midbody, disappeared from this site more rapidly in the non-adherent cells. A FAK-Src signaling pathway downstream of integrin-mediated cell adhesion was found to decelerate both PLK1 degradation and CEP55 accumulation at the midbody. These data identify the regulation of PLK1 and CEP55 as steps where integrins exert control over the cytokinetic abscission.

Previous studies have shown that cytokinetic abscission at the end of mitosis is executed by the ESCRT machinery in mammalian cells, and that the process is dependent on adhesion-induced integrin signalling via a FAK-PLK1-CEP55-TSG101/Alix-CHMP4B pathway. The present study identified an alternative abscission mechanism driven by mechanical force. In the absence of integrin signals (non-adherent conditions), cytokinesis in non-transformed human fibroblasts proceeds to CEP55 accumulation at the midbody, but after prolonged time (>3 hours) the major midbody components Aurora B, MKLP1 and CEP55 were no longer detected in the area. Upon adhesion to fibronectin, such cells were able to complete abscission without re-appearance of midbody proteins. Live-cell imaging revealed that re-plating on stiff fibronectin matrix (64 KPa) allowed >95% of the cells to complete abscission within 9 hours while the corresponding number was 40% on soft fibronectin matrix (0.5 KPa). The cells re-plated on poly-L-lysine were not able to generate tension and did not divide. Thus, mechanical tension can cause cytokinetic abscission by stretching of the intercellular bridge between the two daughter cells until it eventually ruptures without the involvement of ESCRT complexes. Importantly, regression of the cleavage furrow and formation of bi-nucleated cells did not occur in most of the suspension-treated mitotic cells after re-plating on fibronectin. Septin, which stabilizes the membrane associated with the midbody, was found to remain along the ingressed membrane, suggesting that this filament system maintains the membrane bridge although the midbody had dissolved, thereby preventing regression and allowing tension to act on the narrow intercellular bridge.