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

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.


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For initially assessing the kinetics of Arp2/3 complex depletion, DMSO-and 4-OHT-127 treated cells were collected at various timepoints, lysed, and immunoblotted with antibodies to 128 the ArpC2 and Arp3 subunits plus antibodies to GAPDH and tubulin as loading controls (Fig   129   1A). After 1 day in 4-OHT, ArpC2 protein levels were reduced by nearly half compared to 130 DMSO-treated Flox cells (Fig 1A and 1B). By 2 days, ArpC2 expression was diminished by 131 approximately 80%, and a reduction in Arp3 levels became noticeable (Fig 1A and 1B). The 132 amounts of both subunits continued to steadily decline in the iKO cells over time until they were 133 8 absent following 5 days in 4-OHT (Fig 1A and 1B). DMSO and 4-OHT were removed from 134 culture media after 6 days, but ArpC2 and Arp3 remained undetectable in the iKO population 135 out to 13 days (Fig 1A and 1B). To independently confirm the loss of the Arp2/3 complex by 136 fluorescence microscopy, Flox and iKO cells were stained with an antibody to label Arp3 and 137 with fluorescent phalloidin to visualize F-actin. As expected, Flox cells exhibited prominent Arp3 138 staining within F-actin-rich peripheral membrane ruffles, whereas Arp3 staining and ruffles were 139 both missing from the iKO cells ( Fig 1C). Collectively, these results demonstrate that in this 140 cellular context, the Arp2/3 complex knockout is rapid, complete, and stable over time.

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To determine the impact of abolishing Arp2/3 complex expression on cell proliferation, 142 we next quantified cell titers on a daily basis following the addition of DMSO or 4-OHT. For the 143 first 3 days, Flox and iKO cells multiplied at identical rates, but by 4 days, the growth 144 characteristics of Flox and iKO cultures began to diverge, and at 5 days, the iKO samples were 145 proliferating at a clearly slower pace (Fig 1D). After approximately 10-12 days, virtually all iKO 146 cells stopped dividing (Fig 1D). To quantify the differences in cell multiplication rates, we

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When examining the different growth characteristics of the Flox and iKO cultures, it also 9 that iKO cells were flatter and larger than Flox cells (Fig 1F). Quantification of Flox and iKO cell 160 areas showed that the iKO cells were, on average, about twice as large as Flox cells (Fig 1G).

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So in addition to losing their ability to multiply, Arp2/3-deficient cells display significant increases 162 in their size.  Interleukin-6 (Il-6), a pro-inflammatory cytokine consistently present in the SASPs derived from 175 senescent cells with diverse origins [49,50]. In agreement with findings from the ArpC2/Arp2 176 RNAi MEF studies, Il-6 expression was greater in iKO cells than in Flox cells at 3, 6, and 9 days 177 after the onset of 4-OHT treatment (Fig 2A). RT-qPCR revealed that Il-6 transcript levels were 178 nearly 4-fold higher in the iKO compared to Flox cells at 9 days (Fig 2B), indicating that a 179 permanent loss of the Arp2/3 complex induces the production of this key SASP component.

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Senescence is also frequently associated with changes in nuclear structure, including 181 decreased levels of Lamin B1, a structural protein of the nuclear lamina [51]. To determine if 182 Lamin B1 abundance was altered by the deletion of ArpC2, we immunoblotted Flox and iKO 183 cells for Lamin B1 and found that Lamin B1 protein levels were indeed 3-fold lower in iKO cell 184 populations (Fig 2C and 2D). To confirm the reduction in Lamin B1 expression, Flox and iKO 10 cells were also subjected to immunofluorescence microscopy. In Flox cells, nuclear Lamin B1 186 staining was consistently bright (Fig 2E and 2F). Contrastingly, in iKO cells, nuclear Lamin B1 187 levels were visibly lower (Fig 2E and 2F). In agreement with the immunoblotting data, 188 quantification of nuclear fluorescence intensities demonstrated that, on average, Lamin B1 was 189 nearly 3-fold less abundant in iKO cells (Fig 2F).

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In addition to the production of a transcriptional SASP response and a decrease in their 191 nuclear Lamin B1 levels, senescent cells typically exhibit a cytoplasmic senescence-associated  Fig 3C). Quantification of LysoTracker fluorescence at day 9 revealed that more than 201 80% of iKO cells versus 5% of Flox cells showed the more expansive staining pattern ( Fig 3D).

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Micronuclei ranged in size and could be found completely detached (Fig 4B; i, ii, vi) or tethered 218 to the periphery (Fig 4B; iii, iv, v) of the main nucleus. We next quantified the timing and   (Fig 5B), whereas iKO cells frequently had 1-3 isolated γH2AX clusters 231 that localized to the nuclear periphery and/or within micronuclei ( Fig 5B). Quantification of the 232 percentage of cells with γH2AX clusters following the addition of DMSO or 4-OHT revealed that 233 clusters began to increase in iKO cells by 2 days, and that from 3 days onward, clusters were 234 significantly more common in iKO cells than in Flox cells (Fig 5C). The fraction of iKO cells with 235 γH2AX clusters leveled out at approximately 25%, while the proportion of Flox cells containing 236 clusters was always 3-6% ( Fig 5C). These results are consistent with previous observations in 12 which Drosophila and mouse cells exposed to ionizing radiation and subjected to RNAi-238 mediated Arp2/3 depletion were found to contain DNA damage and micronuclei [27]. However,

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our results indicate that even without exposure to acute genotoxic agents, losing the Arp2/3 240 complex can cause an accumulation of damaged DNA elements that incorporate into 241 micronuclei, thereby illustrating that the Arp2/3 complex is a crucial player in maintaining   4-OHT vs DMSO treatment, p53 levels were higher in iKO than in Flox cells, and p53 was 251 phosphorylated on Serine-15 ( Fig 6A). Furthermore, immunofluorescence microscopy revealed 252 that nuclear p53 fluorescence was more intense in iKO cells than in Flox cells ( Fig 6B). These 253 results imply that a p53-mediated DNA damage response is induced in Arp2/3-depleted cells.

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Two of the major factors involved in the cell cycle arrest that leads to senescence are 255 the cyclin-dependent kinase inhibitors Cdkn2a/p16INK4A (p16) and Cdkn1a/p21CIP/WAF (p21). Cdkn1a expression appeared greater in the iKO cells than in the Flox cells at 6 and 9 days after 264 the initiation of 4-OHT treatment ( Fig 6C). RT-qPCR revealed that Cdkn1a transcript levels 265 were doubled in the iKO cells relative to the Flox cells at 9 days ( Fig 6D), showing that deletion 266 of the Arp2/3 complex leads to an upregulation of this key cell cycle regulator. To evaluate 267 whether the increase in Cdkn1a transcript corresponded to greater p21 protein levels, Flox and 268 iKO cells were treated with antibodies to p21 and subjected to immunofluorescence microscopy 269 ( Fig 6E). Quantification of p21 nuclear intensity at the 6 day timepoint verified that p21 levels 270 were significantly higher in iKO than in Flox cells (Fig 6E and 6F). Together, the p53  mCherry-cGAS or mCherry as a control (Fig 7A), and treated them with 4-OHT to induce ArpC2 285 deletion. mCherry was highly expressed in the iKO cells but was not recruited to micronuclei 286 ( Fig 7B). In contrast, mCherry-cGAS showed intense localization to micronuclei (Fig 7B),

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indicating that it can detect the cytosolic DNA in iKO cells. 14 MTFs. Immunoblotting indicated that cGAS and STING were expressed at similar levels in Flox 290 and iKO cells (Fig 7C), so to test whether cGAS and/or STING were recruited to the 291 micronuclei, we visualized these proteins via immunofluorescence microscopy after 3-4 days, 292 when micronuclei become more abundant in iKO cells and just before Flox and iKO 293 multiplication rates begin to diverge. Consistent with previous studies [73,74], endogenous 294 cGAS was present in the nuclei of both Flox and iKO cells ( Fig 7D). It was also readily apparent 295 in nearly 40% of iKO cell micronuclei ( Fig 7D). The rare micronuclei that formed in Flox cells 296 also recruited cGAS (Fig 7D magnification). For STING, an integral membrane protein that

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To explore whether cGAS affects the cell proliferation arrest that takes place when the 306 Arp2/3 complex is lost, MTFs were exposed to 4-OHT for 3 days to induce the deletion of ArpC2

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OHT treatment, when Arp2/3 complex levels were declining the fastest (Fig 1B), the cells were 325 still dividing rapidly (Fig 1E), and the incidence of micronucleus formation was highest ( Fig 4C).

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Timelapse imaging of H2B-GFP-expressing Flox cells revealed that the majority of 327 mitoses resulted in an equal partitioning of nuclear chromatin into two daughter cells (Fig 8A).

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However, iKO cell mitoses often yielded micronuclei due to errors in chromatin segregation (Fig   329   8A). This was repeatedly observed when chromatin fragments near the metaphase plate were 330 not properly distributed to daughters during anaphase (Fig 8A). Another common phenotype of 331 iKO cells that entered mitosis was premature mitotic exit ( Fig 8B). Such cells underwent a 332 prolonged prometaphase or metaphase, failed to enter anaphase, and ultimately returned to 333 interphase with nuclei containing twice their normal chromatin content (Fig 8B). Micronuclei 334 also formed in some of these cells that failed to complete mitosis ( Fig 8B).

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We next measured the frequencies with which the mitotic defects occurred. First, 336 approximately 23% of completed mitoses in iKO cells yielded micronuclei compared to only 337 about 6% of completed mitoses in Flox cells (Fig 8C). Second, after categorizing the stages of 338 mitosis and determining the number of timelapse frames spent specifically in metaphase 339 (judged by the presence of at least 95% of H2B-GFP fluorescence aligned compactly at the cell 340 equator), we found that >80% of Flox cells that completed mitosis spent only one frame in 341 16 metaphase, whereas only 40% of iKO cells proceeded through metaphase with this speed (Fig   342   8D). The other 60% of iKO cells that completed mitosis spent two or more frames in metaphase 343 (Fig 8D), suggesting that iKO cells experience an unusually prolonged metaphase period.

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Third, when evaluating the incidence of premature mitotic exits (defined as cells that entered 345 prophase but did not complete anaphase), we discovered that nearly 30% of iKO cells that       (Fig 9D). This was not due to a general deficit in central spindle staining, 394 because the fluorescence intensity of microtubules in the same region was not significantly 395 different between iKO and Flox cells (Fig 9D). A reduction in the penetration of actin filaments, 396 but not microtubules, into the metaphase chromatin-containing region of iKO cells was further 397 reflected by fewer prominent F-actin peaks in fluorescence intensity plot profiles (Fig 9E).

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Collectively, these results indicate that a functional Arp2/3 complex is required for the   Fig 10B). In contrast, iKO cells displayed unbalanced tubulin intensities in 410 which the microtubules appeared to be more heavily concentrated on one side of the spindle 411 (Fig 10A and 10B). These results suggest that in addition to the presence of misplaced

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The Arp2/3 complex is a key driver of many cellular processes that are mediated by actin 421 assembly at the plasma membrane, namely adhesion, endocytosis, protrusion, and migration.

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While the complex is evolutionarily conserved among nearly all eukaryotes and is essential for

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Despite uncertainties in the severity of cytoplasmic organelle dysfunction in iKO cells, 446 obvious defects in nuclear and chromatin-associated processes emerged as major factors in 447 promoting senescence in our studies. Kinetic analyses indicate that 1-3 days after exposure to 448 4-OHT, ArpC2 (and thus functional Arp2/3 complex) levels are 50-90% depleted (Fig 1A and   449 1B). This time period coincides with the steepest increases in detection of micronuclei via DAPI 450 staining ( Fig 4C) and visualization of dsDNA breaks in both nuclei and micronuclei via γH2AX 451 staining (Fig 5C). A DNA damage response is activated, as evidenced by increased p53 452 expression and phosphorylation (Fig 6A and 6B). By 4-6 days, the p53-regulated cell cycle 453 inhibitor p21 accumulates in the nucleus (Fig 6C-F) and cell multiplication stops (Fig 1D). By 7 454 days, SA-βgal-positivity begins to increase (Fig 3A and 3B). At 7-10 days, the entire population 455 of iKO cells appears to be senescent, given the complete lack of proliferation (Fig 1D), as well 456 as consistent single cell phenotypes in enlargement (Fig 1G), nuclear Lamin B1 reduction (Fig   457   2F), and cytoplasmic acidity (Fig 3D). Beyond 10 days, the iKO cell population remains viable 458 but non-replicative while maintaining 20-25% positivity for micronuclei and SA-βgal activity.

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The main mechanism underlying micronucleus biogenesis in Arp2/3-deficient cells is a 494 lack of fidelity in chromatin partitioning during mitosis, as revealed by our live imaging studies.      For mCherry-cGAS cloning, mouse cGAS was amplified via PCR from a pMSCVpuro-eGFP-557 cGAS template (Addgene, 108675) using primers containing KpnI and NotI restriction sites (S1 558   Table), and inserted into the pKC-mCherryC1 vector [99].

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RNA from Flox and iKO fibroblasts grown in 6-well plates was isolated using TRIzol reagent 571 (Ambion). Following chloroform extraction, isopropanol precipitation, and a 75% ethanol wash, 572 total RNA was resuspended in water. cDNA was reverse transcribed using the iScript cDNA 573 synthesis kit (Bio-Rad) and then PCR-amplified using Taq polymerase (New England Biolabs) 574 and primers listed in S1 Table. Primers were designed to amplify ~340-480bp with each cDNA 575 target. The resulting PCR products were visualized on ethidium-bromide stained agarose gels.

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For immunofluorescence, fibroblasts cultured on glass coverslips in 24-well plates were washed 599 with PBS and fixed using PBS containing 2.5% paraformaldehyde for 30min. Following PBS 600 washes, cells were permeabilized using PBS containing 0.1% Triton X-100 for 2min, washed, 601 and stained with primary antibodies in PBS containing 1% bovine serum albumin, 1% FBS, and 602 0.02% azide for 45-60min as described in S2   Clara-E camera, and a computer running NIS Elements Software. Most images were taken as 611 single epifluorescence slices, whereas 60X images of mCherry-transfected cells (Fig 7) were 612 taken as z-stacks with a 0.3µm step size, and fixed mitotic cells were taken with a 0.3µm (Fig 9) 613 or 0.5µm (Fig 8, Fig 10) step size. Live cell imaging was performed in a 35ºC chamber (Okolab).

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Samples were lysed, subjected to SDS-PAGE, and immunoblotted with antibodies to ArpC2,