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Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence [1]

['Elena L. Haarer', 'Department Of Molecular', 'Cell Biology', 'University Of Connecticut', 'Storrs', 'Connecticut', 'United States Of America', 'Institute For Systems Genomics', 'Corey J. Theodore', 'Shirley Guo']

Date: 2023-02

The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.

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 in mammalian cells–causes DNA damage, genomic instability, mitotic chromosome partitioning defects, 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.

Copyright: © 2023 Haarer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To determine the outcome of Arp2/3 complex ablation on chromatin-associated processes related to cell viability and multiplication, we employed the inducible ArpC2 knockout cell model [ 42 ]. Our findings connect Arp2/3 complex functions in maintaining genomic integrity during interphase and mitosis in normal cells to the biogenesis of micronuclei and induction of cellular senescence pathways when Arp2/3 is inactivated.

Although the effects of transient Arp2/3 depletion or inhibition on chromatin repair are now evident, and several aberrations in chromosome movement have been characterized, the impact of total and permanent Arp2/3 ablation on these processes has not been established. The development of several cellular systems for studying long-term Arp2/3 depletion or deletion has allowed more clear-cut assessments of the requirements for the Arp2/3 complex in a given cellular process [ 14 , 40 – 45 ]. For example, these models have already provided fundamental insights into the function of the Arp2/3 complex in cell migration. Studies using mouse embryonic fibroblasts (MEFs) expressing shRNAs targeting the ArpC2 and Arp2 subunits [ 40 ], embryonic stem cell-derived fibroblasts lacking the ArpC3 subunit [ 14 ], fibroblasts harboring tamoxifen-inducible knockouts of the ArpC2 or Arp3 subunits [ 42 , 43 ], and human neutrophil-like cells depleted of Arp2 [ 44 , 45 ] indicate that the Arp2/3 complex is crucial for cell polarization, lamellipodia formation, and/or directional migration in several environmental contexts.

Apart from their functions in chromatin-associated processes during interphase, actin and its nucleators, especially Formins and the Arp2/3 complex, are increasingly being found to support proper chromosome movements during meiosis and mitosis. In starfish oocytes, after nuclear envelope breakdown, several types of F-actin structures promote chromosome transport and coordinate capture by microtubules [ 30 , 31 ]. Studies in mouse oocytes further show that actin filaments permeate the meiotic microtubule spindles and facilitate proper chromosome congression [ 32 , 33 ]. Chemical inhibition of actin dynamics or genetic inactivation of Formin-2 prevents proper formation of kinetochore microtubules and leads to chromosome alignment and segregation errors [ 33 , 34 ]. Similarly, during mitosis, several actin structures have been shown to interact with and possibly guide microtubule spindle components. Actin filaments that run between the microtubule spindle poles and F-actin fingers that project from the cell cortex into the spindle have been identified in Xenopus epithelial cells [ 35 ]. Centrosomes, which serve as major microtubule nucleation and organizing centers, are also sites of actin assembly [ 36 ]. The Arp2/3 complex localizes to centrosomes in multiple mammalian cell types, and pharmacological inhibition of Arp2/3 results in decreased centrosomal actin levels and impaired mitotic spindle formation [ 37 – 39 ]. Thus, disruption of either actin or Arp2/3 function during meiosis or mitosis can lead to defects in chromosome dynamics, highlighting the actin cytoskeleton as a key player in maintaining genomic integrity during nuclear division.

In contrast to the well-characterized roles of the Arp2/3 complex in protrusion and motility, its functions in nuclear processes are only beginning to emerge. During interphase, all 3 classes of actin nucleators promote nuclear actin filament assembly in response to DNA damaging agents [ 26 – 28 ]. In Drosophila and mammalian cells, Arp2/3-mediated actin polymerization is crucial for repositioning damaged heterochromatin to the nuclear periphery, which enables subsequent DNA repair activities [ 27 , 28 ]. Moreover, depletion of the Arp2/3 complex using RNAi in Drosophila larvae leads to chromosomal abnormalities and genomic instability [ 27 ]. Additional studies in human cells exposed to DNA damaging agents indicate that depletion of the Arp2/3 complex also causes defects in pro-apoptotic signaling [ 29 ].

Many processes that involve plasma membrane dynamics, especially cell adhesion and migration, rely on actin networks assembled by the Arp2/3 complex [ 15 ]. In fact, conditional knockouts in mice indicate that the complex is crucial for maintaining normal tissue architecture, promoting changes in cell shape, and powering cell migration during development [ 14 , 16 – 20 ]. These in vivo results are consistent with molecular and cellular studies of Arp2/3-mediated actin assembly using in vitro systems [ 21 ], dominant negative regulatory proteins [ 22 ], transient RNAi-mediated knockdowns [ 23 ], and pharmacological inhibitors of the complex [ 24 , 25 ].

The actin cytoskeleton consists of dynamic protein polymers that have well-known functions in cell morphogenesis and motility. Globular (G-) actin monomers are present in the cytoplasm and nucleus, and their polymerization into filamentous (F-) actin is driven by proteins called nucleators [ 1 ]. These include actin monomer-oligomerizing proteins, Formin-family nucleation/elongation proteins, and the Arp2/3 complex–a heteroheptameric actin assembly factor that binds to the sides of existing filaments and nucleates new filaments to create branched networks [ 2 ]. The Arp2/3 complex is highly conserved across almost all eukaryotes [ 3 , 4 ] and is required for viability in such organisms; inactivation of genes encoding its subunits prevents growth of S.cerevisiae [ 5 , 6 ] and D.discoideum [ 7 ] and is embryonic lethal in animals including D.melanogaster [ 8 , 9 ], C.elegans [ 10 , 11 ], and M.musculus [ 12 – 14 ]. However, the cellular basis underlying the essential nature of the Arp2/3 complex is not well understood.

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010045

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