Matrix compliance and the regulation of cytokinesis

Abstract

Integrin-mediated cell adhesion to the ECM regulates many physiological processes in part by controlling cell proliferation. It is well established that many normal cells require integrin-mediated adhesion to enter S phase of the cell cycle. Recent evidence indicates that integrins also regulate cytokinesis. Mechanical properties of the ECM can dictate entry into S phase; however, it is not known whether they also can affect the successful completion of cell division. To address this issue, we modulated substrate compliance using fibronectin-coated acrylamide-based hydrogels. Soft and hard substrates were generated with approximate elastic moduli of 1600 and 34,000 Pascals (Pa) respectively. Our results indicate that dermal fibroblasts successfully complete cytokinesis on hard substrates, whereas on soft substrates, a significant number fail and become binucleated. Cytokinesis failure occurs at a step following the formation of the intercellular bridge connecting presumptive daughter cells, suggesting a defect in abscission. Like dermal fibroblasts, mesenchymal stem cells require cell-matrix adhesion for successful cytokinesis. However, in contrast to dermal fibroblasts, they are able to complete cytokinesis on both hard and soft substrates. These results indicate that matrix stiffness regulates the successful completion of cytokinesis, and does so in a cell-type specific manner. To our knowledge, our study is the first to demonstrate that matrix stiffness can affect cytokinesis. Understanding the cell-type specific contribution of matrix compliance to the regulation of cytokinesis will provide new insights important for development, as well as tissue homeostasis and regeneration.

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

“…In their model, the fibroblasts in tumors show altered cell-matrix adhesion, increased migration, and changed mechanics, which might stimulate cancer cells to migrate toward stromal regions that are less dense, and thereby increase the size of a tumor. Moreover, cell-matrix stiffness has been shown to stimulate cytokinesis, which suggests that the increased stiffness of the surrounding fibroblasts can also stimulate the proliferation of cancer cells (37). These ideas are in line with our findings that these RhoA-KO tumor-promoting fibroblasts showed increased homogeneous stiffness and fewer soft locations, with altered cytoskeleton and cell-matrix adhesion.…”

Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication

“…[22][23][24]26 To investigate the fate of binucleated cells induced by loss of substrate adhesion, human fibroblast MRC-5 mitotic cells were isolated by mitotic shake off, maintained in suspension for 30 min to allow mitotic progression, seeded in growth medium and analyzed for several parameters at different time points. Parallel cultures were treated with the microtubule inhibitor nocodazole (NOC) for 3 hrs, then released in drug-free medium for 30 min to allow reformation of the mitotic spindle and subjected to the same procedure ( Figure 1A).…”

“…Concordantly, inhibition of intercellular bridge abscission and fusion of connected daughter cells have been demonstrated by live cell imaging of dermal fibroblasts performing cytokinesis on experimentally generated soft substrates. 26 Binucleated cells produced by cytokinesis in suspension preferentially arrest in the G2 phase…”

Alpha 6 integrins promote cytokinesis by regulating the expression of RSK2 and MKLP1

“…It is well established that the compliance of the adhesion substrate impacts integrin signaling and cytokinesis (23,24). Our studies with SIMS cells were performed on culture plates, whose stiffness is orders of magnitude higher than observed in tissues (25).…”

“…Much of what is known about the mechanisms that regulate cytokinesis in mammalian cells has been acquired from experiments in culture using cell lines that are easily transfected and imaged (22). However, since integrin signaling can be strongly influenced by substrate compliance (23), and our previous studies demonstrated that cytokinesis is regulated by matrix compliance in a cell typedependent manner (24), it is important to demonstrate that pathways that regulate cytokinesis in cell culture similarly regulate cytokinesis in a more physiological complex context. Since we previously demonstrated that α6 integrins promote successful cytokinesis in embryonic salivary glands in ex vivo organ culture (14), we again used this model to test whether RSK2 regulates cytokinesis and MKLP1 expression in this context.…”