Src kinase promotes actin patch formation to prevent chromosome breakage in cytokinesis with chromatin bridges


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Eleni Petsalaki, Sofia Balafouti, George Zachos

Abstract

Background

DNA bridges are strands of incompletely segregated chromatin that connect daughter nuclei. If unresolved, chromatin bridges can break in cytokinesis leading to micronuclei formation and accumulation of DNA damage, which are associated with tumorigenesis. To prevent this, human cells accumulate actin (actin patches) at the base of the intercellular canal that connects the two daughter cells to stabilize chromatin bridges; however, the molecular mechanisms involved are incompletely understood.

Method

To investigate this, we used site directed mutagenesis, RNA silencing, replacement of endogenous proteins with transfected wild-type or mutant transgenes, confocal microscopy, live cell imaging and biochemistry techniques.

Results

We show that Src, a protein-tyrosine kinase that regulates actin dynamics, and Chk1, a kinase of the DNA damage response, are required for actin patch formation in the presence of chromatin bridges in human cell lines. Chk1 phosphorylates human Src-serine 51 (S51) to fully induce Src catalytic activity; in turn, phosphorylated Src-S51 promotes formation of actin patches and prevents chromatin bridge-breakage in cytokinesis. Expression of the nonphosphorylatable mutant Src–GFP-S51A induced chromatin breakage and diminished actin patches. Src- or Chk1-deficient cells exhibited increased frequency of micronuclei that were positive for γ-H2AX staining compared with controls. We also show that RhoA, a member of the Rho family of small GTPases which control the growth or contraction of filamentous actin fibers, localizes to actin patches in control cells. Furthermore, inhibition of RhoA correlates with chromatin breakage and impaired formation of actin patches in the presence of chromatin bridges.

Conclusion

These results show that Chk1, Src and RhoA promote formation of actin patches and prevent chromatin breakage in cytokinesis.

Impact statement

Because cancer cells usually exhibit relatively high levels of DNA replication stress that can lead to DNA bridges, the mechanisms we have identified may be important to prevent tumor formation or progression.