Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence

Abstract The Arp2/3 complex is a ubiquitous actin nucleator with well-characterized activities in cell organization and movement, but its roles in chromatin-associated and cell cycle-related processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity, mitosis, and cell proliferation using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Upon Arp2/3 depletion, cells undergo an abrupt proliferation arrest that is accompanied by activation of the tumor suppressor p53, upregulation of its downstream cell cycle inhibitor Cdkn1a /p21, and recognition of micronuclei by the cytosolic DNA sensor cGAS. Micronuclei arise in ArpC2 iKO cells due to chromosome segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged chromatin fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. These studies establish functional requirements for the mammalian Arp2/3 complex in genome stability and mitotic spindle organization. They further expand our understanding of the intracellular mechanisms that lead to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging. Author Summary The actin cytoskeleton consists of protein polymers that assemble and disassemble to control the organization, shape, and movement of cells. However, relatively little is understood about how the actin cytoskeleton affects genome maintenance, cell multiplication, and biological aging. In this study, we show that knocking out the Arp2/3 complex, a core component of the actin assembly machinery, causes DNA damage, genomic instability, defects in chromosome partitioning during mitosis, and a permanent cell proliferation arrest called senescence. Since senescent cells are major contributors to both age-associated diseases and tumor suppression, our findings open new avenues of investigation into how natural or experimental alterations of cytoskeletal proteins impact the process of aging and the regulation of cancer.