An ATM–Chk2–INCENP pathway activates the abscission checkpoint

How cells delay completion of cytokinesis (abscission) in the presence of chromatin bridges is unclear. Petsalaki and Zachos describe an ATM–Chk2–INCENP signaling pathway that activates the abscission checkpoint to prevent chromatin breakage in cytokinesis.


Introduction
To ensure faithful inheritance of the genetic material during cell division, completion of cytokinesis (abscission) is tightly coordinated with chromosome segregation (Mierzwa and Gerlich, 2014). In response to chromosome segregation defects giving rise to chromatin bridges or lagging chromosomes trapped inside the intercellular canal (Gisselsson, 2008), eukaryotic cells delay abscission to prevent chromatin breakage and tetraploidization by regression of the cleavage furrow (Bai et al., 2020;Carlton et al., 2012;Gisselsson, 2008;Norden et al., 2006;Steigemann et al., 2009;Thoresen et al., 2014), which are associated with genomic instability and cancer predisposition (Ganem and Pellman, 2012;Lens and Medema, 2019;Sadler et al., 2018). In mammalian cells, this abscission delay is called the "abscission checkpoint" and is dependent on Aurora B kinase activity at the midbody Zachos, 2016, 2019;Steigemann et al., 2009).
The activity and targeting of Aurora B depend on its partners in the chromosomal passenger complex (CPC) that also includes the scaffolding protein INCENP and the nonenzymatic subunits Survivin and Borealin (Carmena et al., 2012;Honda et al., 2003). In human cells, CPC localization to central spindle microtubules requires INCENP binding to Mklp2 kinesin (Adriaans et al., 2020;Gruneberg et al., 2004;Hümmer and Mayer, 2009;Kitagawa et al., 2014;van der Horst et al., 2015). At the midbody, the CPC localizes to the midbody arms (Cooke et al., 1987;Hu et al., 2012;Vagnarelli and Earnshaw, 2004). More recently, a relatively small population of Aurora B was detected inside the Flemming body (FB), the narrow region at the midbody center where tubulin staining by immunofluorescence is blocked (Hu et al., 2012), in late cytokinesis (Petsalaki and Zachos, 2016); however, the significance of this localization for the abscission delay is incompletely understood. Cdc-like kinases (Clk's) 1, 2, and 4 phosphorylate Aurora B serine 331 (Ser331) to fully induce Aurora B catalytic activity at the midbody (Petsalaki and Zachos, 2016). Active Aurora B imposes the abscission checkpoint by phosphorylating the endosomal sorting complex required for transport III (ESCRT-III) subunit charged multivesicular body protein 4C (Chmp4c) to target Chmp4c to the midbody center (Capalbo et al., 2012;Carlton et al., 2012;Petsalaki and Zachos, 2016) to inhibit the ATPase Vps4 and prevent its activity on ESCRT-III filaments thereby delaying abscission (Caballe et al., 2015;Mierzwa et al., 2017;Thoresen et al., 2014). Inhibition of Aurora B also accelerates abscission in normally segregating cells, i.e., in the absence of trapped chromatin, indicating the abscission checkpoint functions more generally as an abscission timer (Carlton et al., 2012;Petsalaki and Zachos, 2016;Steigemann et al., 2009). However, the molecular mechanisms that relay cytoplasmic stresses, such as chromatin bridges, to the CPC have not been previously identified.
The ataxia-telangiectasia mutated (ATM) and Chk2 kinases are master regulators of the DNA damage response (Smith et al., 2010). ATM activation requires the MRN (Mre11-Rad50-Nbs1) double-strand break sensor complex that processes DNA ends and recruits ATM to broken DNA molecules (Bakkenist and Kastan, 2003;Falck et al., 2005;Lee and Paull, 2005;Uziel et al., 2003). In turn, active ATM phosphorylates Chk2 threonine 68 (Thr68) to promote Chk2 activation and stimulate downstream responses (Ahn et al., 2000(Ahn et al., , 2002. In the absence of DNA damage, ATM and Chk2 are required for spindle checkpoint signaling (Petsalaki and Zachos, 2014;Yang et al., 2011). ATM and Chk2 also localize to the midbody in cytokinesis (Tsvetkov et al., 2003;Yang et al., 2011); however, a role for ATM or Chk2 in the abscission checkpoint has not been previously reported. In this study, we show that ATM phosphorylates and activates Chk2 at the midbody center in late cytokinesis in normally segregating cells. In turn, active Chk2 phosphorylates human INCENP at the newly identified site Ser91 to promote INCENP binding to Mklp2, resulting in recruitment of the INCENP-Mklp2 complex to the midbody center through Mklp2's interaction with the midbody protein Cep55 to delay abscission. We also show that in the presence of a chromatin bridge, the MRN complex activates ATM and the downstream Chk2-INCENP signaling pathway at the midbody to delay abscission and prevent chromatin bridge breakage in cytokinesis.

Chk2 inhibition accelerates midbody disassembly
To investigate a role for Chk2 in midbody resolution, HeLa cells stably expressing α-tubulin fused to GFP (tubulin:GFP) were monitored by time-lapse microscopy and the kinetics of tubulin disassembly at the midbody determined. In control cells, the midbody microtubules remained visible for a median time of 43 ± 7 min after formation. In contrast, treatment of cells in cytokinesis with 10 µM Chk2 inhibitor II accelerated midbody disassembly (t = 22 ± 6 min) compared with controls (P < 0.001; Fig. 1, A and B; Video 1; and Video 2). Furthermore, Chk2deficient cells exhibited faster cleavage of the intercellular canal compared with controls by phase-contrast live-cell imaging, indicating premature abscission (P < 0.001; Fig. S1, A and B; Video 3; and Video 4). These findings correlated with reduced frequency of human colon carcinoma BE cells at midbody stage (midbody index) after Chk2 inhibition or Chk2 depletion by siRNA (siChk2), but not with an increase in binucleate/multinucleate or prometaphase cells, suggesting Chk2 inhibition does not prevent mitotic entry and that Chk2-deficient cells can progress through abscission and disassemble their midbodies more rapidly than controls (Fig. 1 C;and Fig. S1, C-E). Depletion of INCENP by an siRNA located in the 39 untranslated region of INCENP (siINCENP) also reduced the frequency of cells at midbody stage compared with controls transfected with negative siRNA; furthermore, simultaneous depletion of Chk2 and INCENP did not further diminish the midbody index, suggesting Chk2 and INCENP regulate abscission timing by acting through the same pathway (Fig. 1 C;and Fig. S1,C and E). We propose that Chk2 regulates proper abscission timing in normally segregating cells.
Chk2 is required for CPC localization to the midbody center During cytokinesis, the microtubule bundles at the midbody progressively get narrower (Carlton et al., 2012;Elia et al., 2011), and midbody thickness ranges from ∼400 to 1,400 nm in BE cells (Petsalaki and Zachos, 2016). In relative "late" midbodies exhibiting midbody thickness of 400-700 nm, the CPC proteins INCENP, Aurora B, Borealin, and Survivin mainly localize to the midbody arms; however, a relatively small population of CPC proteins was also detectable at the midbody center (FB) in control cells (Fig. 1, D, E, G, I, and K). Depletion of Chk2 diminished localization of CPC proteins to the midbody center in late midbodies (Fig. 1, E-L); however, Chk2 depletion did not reduce the total levels of CPC proteins and did not impair localization of the CPC proteins on the midbody arms in relatively early midbodies with midbody thickness of 800-1,400 nm (Fig. S1, F-J). Also, inhibition of Chk2 by Chk2 inhibitor II in cytokinesis after release of cells from a nocodazole-induced prometaphase block diminished INCENP localization to the midbody center in late midbodies (Fig. 1,M and N). Localization of phosphorylated Aurora B-Ser331 (active Aurora B; Petsalaki et al., 2011) or Chmp4c fused to GFP (Chmp4c:GFP) to the midbody center (which correlates with Aurora B catalytic activity at the midbody; Petsalaki and Zachos, 2016) was impaired in Chk2deficient cells compared with controls; furthermore, phosphorylated Aurora B-Ser331 or Chmp4c:GFP mislocalized on the midbody arms in late midbodies (Fig. 2,A and B;and Fig. S1,K and L). Expression of GFP:Chk2 R resistant to degradation by a second Chk2 siRNA (siChk2-2), but not GFP only, rescued INCENP localization to the midbody center and restored the frequency of cells at midbody stage after Chk2 depletion compared with controls (Fig. 2,and Fig. S1,M and N). We propose that Chk2 is required for CPC localization to the midbody center and optimal localization of Chmp4c to late midbodies.
INCENP targeting to the midbody center delays midbody resolution in Chk2-deficient cells To investigate the significance of CPC localization inside the FB for abscission timing, a truncated human INCENP protein (amino acids 49-918) fused to the C-terminal region (amino acids 456-858) of Mklp1 and GFP, GFP:INCENP(FB), was constructed. GFP:INCENP(FB) localizes to the midbody center through its Mklp1 sequence (Hu et al., 2012) but exhibits relatively diminished localization to the midbody arms, because it lacks the centromere-targeting domain of INCENP (amino acids 1-48) that binds to Mklp2 (  Cells were untreated (control) or treated with 10 µM Chk2 inhibitor II immediately before filming. Midbodies are shown by solid arrows. Time is from midbody formation to midbody disassembly (dotted arrows). Related to Video 1 and Video 2. (C) Frequency of midbody stage cells. BE cells were transfected as indicated, or treated with 10 µM Chk2 inhibitor II (inhII) for 4 h. Values represent mean ± SD from three independent experiments (n > 900). ***, P < 0.001 (ANOVA and Student's t test). (D) Midbody cartoon. MTs, microtubules. (E-L) Localization and mean intensity of CPC proteins at the midbody center in BE cells. Individual image planes show 3.5× magnification of the midbodies. (M and N) INCENP localization and mean intensity after treatment of BE cells with 10 µM Chk2 inhII in cytokinesis. Values represent mean ± SD from n cells. Values in control were set to 1. ***, P < 0.001 (Student's t test). Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. Scale bars, 5 µm. GFP:INCENP(FB), but not GFP only, rescued the frequency of cells at midbody stage after Chk2 depletion (Fig. 2 I and Fig. S1 N). Furthermore, Chk2-deficient cells expressing GFP:IN-CENP(FB) exhibited total and phosphorylated Ser331 (active) Aurora B at the midbody center similar to controls (Fig. 2,J and L;and Fig. S2,. In contrast, treatment of GFP:INCENP(FB) cells with 1 µM TG003, an inhibitor of Clk catalytic activity, reduced the frequency of cells at midbody stage and diminished localization of phosphorylated Ser331, but not total, Aurora B at the midbody center compared with controls, indicating Chk2 regulates midbody disassembly independently of Aurora B activation by Clk (Fig. 2, Fig. S1 N;and Fig. S2,. These results suggest that INCENP targeting to the midbody center by Chk2 is required for proper abscission timing in normally segregating cells.

F).
Chk2 is required for INCENP-Ser91 phosphorylation inside the FB To investigate INCENP-Ser91 phosphorylation in cultured cells, an anti-phospho-INCENP-Ser91 antiserum was raised against the human protein sequence. Phosphorylated INCENP-Ser91 (pINCENP-Ser91) localized to the midzone in anaphase, on the midbody arms in early midbodies, and to the midbody center in late midbodies in control cells but was undetectable in interphase, prometaphase, or metaphase or after treatment of cells with the DNA damage agent etoposide (Fig. 3,G,H,and J;and Fig. S2 I). Phosphorylated Ser91 staining was impaired after incubation of the anti-pINCENP-Ser91 antiserum with the phosphorylated (phospho-Ser91) peptide compared with the unphosphorylated (Ser91) synthetic peptide by immunofluorescence, showing this reagent is specific for the phosphorylation (Fig. S2, J-L). Depletion of INCENP diminished phospho-Ser91 staining compared with control cells (Fig. 3, J and K). Also, depletion of Chk2 by siRNA or inhibition of Chk2 by Chk2 inhibitor II in cytokinesis after release of cells from a nocodazole block reduced pINCENP-Ser91 staining inside the FB and associated with mislocalized pINCENP-Ser91 on the midbody arms in late midbodies (Fig. 3, J-M). However, Chk2 was dispensable for Ser91 phosphorylation on the midbody arms in early midbodies, suggesting a different kinase phosphorylates INCENP-Ser91 in early cytokinesis (Fig. 3,H and I). We propose that Chk2 phosphorylates INCENP-Ser91 at the midbody center in late cytokinesis.
Ser91 phosphorylation is required for INCENP localization to the midbody, proper midbody resolution timing, and cell proliferation To investigate the significance of INCENP-Ser91 phosphorylation, BE cells transiently expressing V5/His-tagged, siRNAresistant versions of WT, nonphosphorylatable Ser91 to alanine (Ser91A) or a phosphomimetic Ser91 to aspartic acid (Ser91D) mutant INCENP were analyzed after depletion of the endogenous protein by siRNA (Fig. S2 M). The nonphosphorylatable Ser91A V5/His-INCENP exhibited impaired localization to early or late midbodies compared with the WT protein ( . We propose that INCENP-Ser91 phosphorylation by Chk2 is required for INCENP localization to the midbody center and proper abscission timing in normally segregating cells. We also propose that Ser91 phosphorylation promotes cell proliferation.

Ser91 phosphorylation promotes INCENP binding to Mklp2
INCENP binding to Mklp2 is required for CPC and Mklp2 localization to the midbody (Hümmer and Mayer, 2009). The nonphosphorylatable V5/His-INCENP-Ser91A exhibited reduced association with the endogenous Mklp2 compared with WT or phosphomimetic Ser91D V5/His-INCENP by coimmunoprecipitation experiments in cytokinesis-enriched extracts (Fig. 4 G). Also, recombinant Mklp2 generated by in vitro transcription-translation exhibited reduced binding to purified Ser91A compared with WT or Ser91D GST-INCENP (1-120) by GST pull-down (Fig. 4 H). Chk2-depleted cells exhibited mislocalized Mklp2 on the midbody arms and reduced Mklp2staining at the midbody center in late midbodies compared with controls (   levels of GFP-associated Aurora B, Survivin, and Borealin and similar histone H3-serine 10 phosphorylation (pH3-Ser10; a marker of Aurora B catalytic activity) by GFP-Trap-kinase assay using recombinant histone H3 as substrate (Fig. 5 A and Fig. S3 E). These results show that Ser91 phosphorylation is required for optimal INCENP binding to Mklp2, but not for CPC formation or Aurora B catalytic activity.
INCENP-Mklp2 localizes to the midbody center through Mklp2 binding to Cep55 Because CPC localization inside the FB is required for proper abscission timing, we investigated the mechanism by which the INCENP-Mklp2 complex localizes to the midbody center. The midbody adaptor protein Cep55 also localizes inside the FB (Zhao et al., 2006); furthermore, Cep55 fused to GFP (Cep55: GFP) colocalizes with INCENP at the midbody center in late midbodies (Fig. 5 B). Depletion of Cep55 impaired INCENP or Mklp2 localization to the midbody center in late midbodies (

ATM inhibition accelerates midbody resolution
Total ATM and phosphorylated ATM-Ser1981 (active ATM; Bakkenist and Kastan, 2003) localized to the midbody center in late midbodies in control cells (Fig. 6 E and Fig. 7 K). Depletion of ATM abolished phospho-ATM-Ser1981 staining by immunofluorescence, confirming the antibody is specific (Fig. S3, L and M). Treatment of HeLa tubulin:GFP cells in cytokinesis with 10 µM KU-55933, an ATM kinase inhibitor, accelerated midbody disassembly compared with controls ( Fig. 6, F and G). Furthermore, HeLa tubulin:GFP cells treated with KU-55933 in cytokinesis exhibited faster cleavage of the intercellular canal compared with controls by phase-contrast live-cell imaging, indicating premature abscission (P < 0.001; Fig. S3,N and O;and Video 5). Also, BE cells exhibited reduced frequency of cells at midbody stage after ATM depletion compared with controls, and the frequency of bi/multinucleate or prometaphase cells was similar in both treatments (Fig. 6 H and Fig. S3 P). These results suggest that ATM catalytic activity delays abscission in normally segregating cells.
ATM is required for Chk2 activation at late midbodies Phosphorylation of Thr68 is required for Chk2 activation (Ahn et al., 2000(Ahn et al., , 2002. Depletion of ATM by siRNA or inhibition of ATM by KU-55933 in cytokinesis after release of cells from a nocodazole block, diminished localization of phosphorylated Chk2-Thr68, total INCENP, total Aurora B, or phosphorylated INCENP-Ser91 to the midbody center compared with controls in late midbodies ( Aurora B, but not Mre11, is required for ATM activation in normally segregating cells Mre11 was not detected at late midbodies; furthermore, Mre11 depletion did not reduce ATM-Ser1981 phosphorylation, suggesting the MRN complex is dispensable for ATM activation (Fig. 7,. In contrast, Aurora B depletion diminished localization of phosphorylated ATM-Ser1981 and Chk2-Thr68, but not total ATM, to late midbodies ( Fig. 7, I-M), suggesting Aurora B is required for ATM activation at the midbody in normally segregating cells (Fig. 7 D, dashed arrow).

Chk2 prevents chromatin breakage in cytokinesis
We next investigated a potential role of Chk2 in cytokinesis with chromatin bridges. Chk2-deficient cells exhibited increased frequency of broken chromatin bridges compared with controls as judged by DNA or γ-H2AX staining; furthermore, simultaneous depletion of Chk2 and INCENP did not exacerbate broken chromatin bridges in Chk2-deficient cells (Fig. 8, A-D). Inhibition of the spindle checkpoint kinase Mps1 by 10 µM AZ3146 that fully inhibits Mps1 catalytic activity did not induce broken chromatin bridges (Fig. 8 B), suggesting inhibition of the mitotic spindle checkpoint does not lead to defective activation of the abscission checkpoint (Abrieu et al., 2001;Petsalaki and Zachos, 2014).
To further investigate the role of Chk2 in chromatin breakage, HeLa cells expressing the inner nuclear envelope marker    LAP2b fused to RFP (LAP2b:RFP) that correlates with chromatin bridges (Steigemann et al., 2009) and displaying LAP2b:RFP bridges in cytokinesis were monitored for up to 180 min by time-lapse microscopy ( Fig. 8 E). We found that 20 of 20 control cells exhibiting intercellular canals with LAP2b:RFP bridges sustained those canals and the LAP2b:RFP bridges for the duration of the experiment. In contrast, 18 of 28 cells (64%) treated with Chk2 inhibitor II in cytokinesis exhibited breakage of LAP2b:RFP-positive intercellular canals and LAP2b:RFP bridges after ∼52-62 min (n = 18; Fig. 8, E and F; Fig. S4, A and C; and Video 6, Video 7, and Video 8). Consistently, Chk2-deficient HeLa LAP2b:RFP cells exhibited increased frequency of broken LAP2b:RFP bridges compared with controls by confocal microscopy analysis of fixed samples (Fig. 8, G and H). Expression of dominant-negative mutant GFP:Vps4-K173Q that inhibits abscission (Morita et al., 2007) diminished broken chromatin bridges after Chk2 depletion compared with GFP-only controls in BE cells by confocal microscopy analysis of fixed samples (Fig. 8 I;and Fig. S4,D and E). Also, Chk2 was not required for formation of actin patches at the bases of the intercellular canal that stabilize chromatin bridges (Fig. S4, F-H; Dandoulaki et al., 2018). We propose that Chk2 is required for the abscission checkpoint in cytokinesis with chromatin bridges. ATM activates Chk2 at the midbody in cytokinesis with chromatin bridges Phosphorylated (active) Chk2-Thr68 or Chk2-Thr383, total ATM, or phosphorylated (active) ATM-Ser1981 localized to the midbody in control cells with chromatin bridges in the absence of double-strand DNA breaks as evidenced by lack of γ-H2AX staining (Fig. 9, L-O). Depletion of ATM diminished localization of phosphorylated Chk2-Thr68 or total INCENP to the midbody and increased broken chromatin bridges compared with control cells (Fig. 9, O and P; and Fig. 10, A, C, and K). Also, 20 of 30 HeLa LAP2b:RFP cells (67%) exhibited breakage of LAP2b:RFP-positive intercellular canals and LAP2b:RFP bridges after treatment with the ATM inhibitor KU-55933 in cytokinesis by live-cell imaging (Fig. 10, D and E; Fig. S4, A-C; and Video 9 and Video 10). These results suggest that ATM is required for Chk2 activation and stable chromatin bridges in cytokinesis.

The MRN complex is required for ATM activation in cytokinesis with chromatin bridges
The MRN complex proteins Mre11, Rad50, and Nbs1 localized to the midbody in BE control cells in cytokinesis with chromatin bridges; furthermore, depletion of any single Mre11, Rad50, or Nbs1 protein diminished localization of the remaining two MRN proteins at the midbody, suggesting MRN proteins localize to the midbody in an interdependent manner (Fig. 10, F and G; Fig. S4, O-R; and Fig. S5, A and B; also compare with the Mre11 intensity at the midbody in normally segregating control cells). Depletion of Mre11 or Rad50 reduced localization of phosphorylated Chk2-Thr68, phosphorylated ATM-Ser1981, phospho-Aurora B-Ser331, or total INCENP at the midbody and increased the frequency of broken chromatin bridges compared with control cells (Fig. 9, O and P; Fig. 10, B, C, and H-K; and Fig. S5, C and D). Furthermore, after depletion of the endogenous Nbs1, expression of siRNA-resistant GFP:Nbs1 (ΔC20) lacking the C-terminal 20 amino acids of human Nbs1 that are required for ATM binding (Falck et al., 2005) diminished localization of total ATM or INCENP to the midbody and increased chromatin breakage compared with cells expressing WT GFP:Nbs1 (Fig. S5, E-J). Depletion of Aurora B by siRNA or treatment of cells with 25 µM mirin, an inhibitor of Mre11 nuclease activity that is required for DNA break processing, did not reduce ATM-Ser1981 phosphorylation at the midbody compared with control cells (Fig. 10

Discussion
In response to chromatin bridges in cytokinesis, the CPC localizes to the midbody to delay abscission to prevent chromatin breakage or tetraploidization (Carlton et al., 2012;Petsalaki and Zachos, 2016;Steigemann et al., 2009;Thoresen et al., 2014). However, the molecular mechanisms that relay chromatin bridges to the CPC have not been previously described. In this study, we show that the DNA damage kinases ATM and Chk2 delay abscission by promoting CPC midbody localization in late cytokinesis. More specifically, in the absence of missegregated chromatin, ATM phosphorylates and activates Chk2 at the midbody center. In turn, active Chk2 phosphorylates INCENP-Ser91 to promote INCENP binding to Mklp2 kinesin and localization of the CPC-Mklp2 complex to the midbody center to delay abscission. Inhibition of Chk2 impairs CPC or Mklp2 localization inside the FB in late midbodies; furthermore, expression of nonphosphorylatable INCENP-Ser91A that does not efficiently bind to Mklp2 impairs INCENP or Mklp2 localization to the midbody arms and the midbody center in early or late midbodies. These results are consistent with previous reports that CPC and Mklp2 mutually depend on each other for midzone/midbody localization by Mklp2 transporting CPC along central spindle microtubules and CPC binding promoting the microtubule processivity of Mklp2 in cytokinesis (Adriaans et al., 2020;Hümmer and Mayer, 2009;Kitagawa et al., 2013;Serena et al., 2020). Of note, however, INCENP binding to Mklp2 is not required for Mklp2's interaction with the midbody protein Cep55 by GST pull-downs when proteins can freely interact in cell extracts, indicating the CPC-Mklp2 complex localizes to the midbody center through Mklp2 binding to Cep55. Deletion of human Mklp2 (amino acids 800-890) C-terminal region diminishes the Mklp2-Cep55 interaction in cell extracts and in vitro and impairs INCENP localization to the midbody center. Because Mklp2 (amino acids 800-890) overlaps with a myosin-II binding site (Fig. S3 J; Kitagawa et al., 2013), a potential role for myosin-II in stabilizing the Mklp2-Cep55 interaction in cell extracts cannot be formally excluded and requires further investigation. Also, because the CPC and Mklp2 are interdependent for their localization to the midbody, a direct role for Mklp2 in the abscission delay, beyond targeting of the CPC to the midbody, cannot be excluded (Fung et al., 2017). However, because expression of midbody-targeted GFP:INCENP(FB) rescues the frequency of cells at midbody stage after Chk2 depletion and GFP:INCENP(FB) exhibits impaired binding to Mklp2, we believe accelerated abscission in Chk2-deficient cells can be attributed to impaired CPC localization to the midbody center.
Phosphorylated INCENP-Ser91 localizes to the midzone in anaphase, on the midbody arms in early midbodies, and inside the FB in late midbodies. Chk2-deficient cells exhibit impaired Ser91 phosphorylation inside the FB; however, Chk2 is dispensable for Ser91 phosphorylation on the midbody arms, suggesting a different kinase phosphorylates INCENP-Ser91 in early midbodies. Because Chk2 localizes to the midbody center in both early and late midbodies, this division of labor likely reflects different intracellular compartments in which the Ser91targeting kinases localize and/or are activated. This kinase promiscuity is perhaps reminiscent of the Aurora B-Ser331 phosphorylation at kinetochores by two different kinases (Chk2 and Chk1) in, respectively, early or late prometaphase (Petsalaki et al., 2011;Petsalaki and Zachos, 2014). It also implies that Ser91 undergoes a rapid phospho-cycling, perhaps with the involvement of a phosphatase. Recruitment of phosphatases to the midbody during abscission has been previously reported (Bhowmick et al., 2019;Fung et al., 2017), and this notion is consistent with our using of the phosphatase inhibitor microcystin during cell fixation to visualize Ser91 phosphorylation by immunofluorescence (please also see Materials and methods).
We also show that Aurora B is required for ATM and Chk2 activation at the midbody in normally segregating cells. Our results suggest that Aurora B catalytic activity is required for the interdependent Mklp2 and CPC recruitment to the midbody (Kitagawa et al., 2013) by governing Chk2 activation, thus imposing INCENP-Ser91 phosphorylation at the FB. Also, these findings are in agreement with previous data showing that active ATM phosphorylated by Aurora B localizes to the midbody where it colocalizes with Aurora B (Yang et al., 2011).
In contrast, in cytokinesis with chromatin bridges, the MRN complex is required for activation of the ATM-Chk2-INCENP pathway at the midbody to delay abscission and prevent chromatin breakage. It should perhaps be noted that Steigemann et al. (2009) had observed binucleation instead of chromatin breakage after Aurora B inhibition by chemical inhibitors using the same HeLa LAP2b:RFP cell line. Because Chk2 regulates CPC localization to the midbody center in late cytokinesis, but not in early midbodies, one possibility is that potent Aurora B inhibition at relatively early midbodies with chromatin bridges in the study by Steigemann et al. (2009) destabilizes the anchoring of the plasma membrane to the midbody, leading to binucleation, whereas impaired CPC localization to the midbody remnant in late midbodies after Chk2 inhibition in our study leads to premature abscission and chromatin breakage. Better understanding of the midbody morphology in cytokinesis with chromatin bridges is required to address these possibilities.
Inhibition of ATM or Chk2 did not accelerate midbody resolution in normally segregating cells in a previous study based on live-cell imaging of a HeLa cell line (Mackay and Ullman, 2015). This appears to be at odds with findings from our study showing that ATM or Chk2 inhibition, or expression of a nonphosphorylatable Ser91A INCENP, accelerates abscission compared with control cells, as judged by timing of midbody disassembly and cleavage of the intercellular canal in HeLa and BE cells by time-lapse microscopy and also by midbody index analysis of fixed cells. Although the reason for the difference between the results of Mackay and Ullman (2015) and our study is unknown, it could be due to genomic differences in the cell lines used resulting in varying levels of ATM or Chk2 inhibition (Liu et al., 2019b).
Our findings are the first to describe an MRN-ATM-Chk2-INCENP pathway that activates the abscission checkpoint by signaling chromatin bridges to the CPC and identify the molecular mechanism by which the CPC localizes to the midbody in cytokinesis with DNA bridges through Mklp2mediated association with Cep55. Our findings also highlight a previously undescribed mechanism by which ATM and Chk2 could protect against genomic instability and tumorigenesis (Antoni et al., 2007;Smith et al., 2010) through their role in abscission checkpoint signaling, and we propose that the abscission checkpoint machinery and DNA damage pathways crosstalk to maintain genome integrity (Petsalaki and Zachos, 2020). Although unchecked ATM activity on DNA after DNA repair may lead to cytokinesis defects (Liu et al., 2019a), our results show that localized ATM activity on the midbody is required for proper abscission timing in the absence of DNA damage. Also, the INCENP-Ser91 consensus site is conserved only in mammals, suggesting that this mechanism might have emerged late in evolution and indicating potential differences between the mechanisms for chromosome segregation sensing in higher eukaryotes and the budding yeast, where the CPC can activate the abscission delay ("NoCut") by physically binding to acetylated chromatin at the spindle midzone (Mendoza et al., 2009).
How Aurora B (which mainly localizes to the midbody arms) sustains formation of an abscission-control protein complex inside the midbody center in normally segregating cells has not been fully understood (Carlton et al., 2012;Petsalaki and Zachos, 2019;Thoresen et al., 2014). Our observations that a relatively small population of CPC molecules localizes to the midbody center in late cytokinesis and that this localization is required for the abscission delay provide an apparent explanation and are in agreement with previous findings that Aurora B phosphorylation by Clk inside the FB is required for the abscission delay (Petsalaki and Zachos, 2016).
The morphology of the midbody appears different in the presence of a chromatin bridge compared with "unperturbed" midbodies in normally segregating cells, based for example on CPC proteins or Mklp2 colocalizing with Mklp1 as a single dot on the midbody remnant and not exhibiting "midbody arms" localization in control cells with chromatin bridges. One possibility is that in cytokinesis with chromatin bridges exhibiting a relatively long and narrow intercellular canal, midbody arms/ microtubules are quickly disassembled, giving rise to midbody remnants that correspond to the midbody center/FB (Connell et al., 2009). Although a more systematic analysis of the midbody structure in the presence of chromatin bridges is required, our results are consistent with the midbody pool of ATM, Chk2, Mklp2, or CPC proteins in the presence of a chromatin bridge corresponding to the "midbody center/FB" identified in unperturbed late midbodies. Such differences in midbody structure may also explain why the mislocalization of the FB pool of phosphorylated Aurora B Ser331 or phospho-INCENP-Ser91 on the midbody arms after Chk2 inhibition that is observed in unperturbed late midbodies is not apparent in the presence of a chromatin bridge.
It is perhaps striking that ATM can be activated in two different ways at the midbody, by the MRN complex or Aurora B kinase, depending on whether there is a chromatin bridge inside the intercellular canal or not. Because there is no pool of midbody arms/CPC adjacent to the midbody remnant in the presence of a chromatin bridge as discussed in the previous paragraph, a different signal (generated by the MRN complex) may be required to control recruitment of the checkpoint machinery to the FB compared with the unperturbed midbody. Our results argue that the Aurora B-ATM-Chk2-INCENP-Chmp4c pathway functions as an abscission timer (as it does not function in the presence of a chromatin bridge), whereas the MRN-ATM-Chk2-INCENP-Chmp4c pathway, activated in the presence of a bridge, generates a robust abscission delay signal and constitutes the actual checkpoint.
A more detailed mechanism by which ATM is activated at the midbody requires further investigation. Aurora B activates ATM in mitosis through ATM-Ser1403 phosphorylation (Yang et al., 2011). Therefore, one possibility is that midbody proteins function as scaffold to promote or regulate the Aurora B-ATM interaction in cytokinesis in normally segregating cells. How does the MRN activate ATM in cytokinesis with chromatin bridges? It was previously reported that continuous immobilization of MRN components to chromatin can activate the ATM-Chk2 signaling pathway in the absence of damaged DNA (Soutoglou and Misteli, 2008). One possibility is that chromatin remodeling enzymes acting to resolve the intertwined DNA strands can promote stable binding of the MRN complex to the chromatin bridge and subsequent ATM activation at the midbody (Chan et al., 2007).
Whether or how the abscission checkpoint signaling can be exploited to improve cancer therapy has remained an open question, at least in part because inhibition of abscission-control proteins such as Aurora B or Chmp4c also impairs important cell functions in early mitosis (Petsalaki et al., 2018a(Petsalaki et al., , 2018bSadler et al., 2018). Because expression of nonphosphorylatable INCENP-Ser91A increases cell death and diminishes cell proliferation and Ser91 is only phosphorylated in cytokinesis, our results support targeting abscission-control proteins as a potential therapeutic strategy to kill proliferating tumor cells (Petsalaki and Zachos, 2019). Chk2-deficient cells exhibit relatively mild proliferation defects compared with cells expressing INCENP-Ser91A (Rainey et al., 2008); however, this could be due to Chk2 regulating Ser91 phosphorylation only in the late stages of cytokinesis. Also, because Chk2 is not an essential gene and Chk2-knockout mice develop normally (Takai et al., 2002), it would be interesting to examine whether Chk2 is required for the abscission checkpoint only in somatic cells and whether the abscission checkpoint is wired differently in embryonic cells. In conclusion, our study describes a molecular mechanism that prevents chromatin breakage in cytokinesis by imposing the abscission checkpoint in human cells by regulating CPC midbody localization.

Materials and methods
Antibodies Anti-pSer91 polyclonal antiserum was generated in rabbits by immunization against the phosphorylated peptide phospho-Ser91 (LSRRK{pS}RSSQLSSR) of human INCENP (Proteogenix). The unphosphorylated peptide Ser91 (LSRRKSRSSQLSSR) was used in competition experiments as appropriate.
Plasmid pEGFPC1/Cep55 encoding human Cep55 into pEGFP-C1 (Clontech) was from Chi-Kuo Hu (Stanford University, Stanford, CA). To generate pGEX4T1/Cep55 plasmids encoding GST-tagged fragments of human Cep55, Cep55 cDNA sequences were amplified by PCR by using pEGFPC1/Cep55 plasmid as template and cloned into the pGEX4T1 vector (GE Healthcare) as EcoRI-XhoI fragments. Plasmid EGFPx2-N1-Nbs1 encoding GFPtagged human Nbs1 was a gift from Jiri Lukas (University of Copenhagen, Copenhagen, Denmark). Briefly, FL Nbs1 was cloned into the XhoI site of EGFP-N1 (Clontech), and an additional GFP was amplified by PCR and cloned into the BamHI site.

Mutagenesis
Point mutations were generated by using the Q5 site-directed mutagenesis kit (New England Biolabs). To generate an siRNAresistant form of Chk2:GFP, the pEGFPN1/Chk2 plasmid coding for human Chk2:GFP was used to introduce C483T, A486G and C489T point mutations giving resistance to the Chk2-2 siRNA. To generate pcDNA3.1/V5-INCENP-Ser91A, the pcDNA3.1/V5-IN-CENP-WT plasmid was used to introduce a T217G point mutation changing Ser91 to alanine. To generate pcDNA3.1/V5-INCENP-Ser91D, the pcDNA3.1/V5-INCENP-WT plasmid was used to introduce T217G and C272A point mutations changing Ser91 to aspartic acid. To generate EGFPx2-N1-Nbs1(ΔC20) encoding GFP: Nbs1 (ΔC20), the EGFPx2-N1-Nbs1 plasmid was used as template to delete nucleotides 2,206-2,265 of human Nbs1 by using the Q5 site-directed mutagenesis kit. Bacteria culture and GST protein purification BL21 (DE3) cells (Agilent Technologies) were grown in 100-200 ml LB Broth (1% wt/vol tryptone, 0.5% wt/vol yeast extract, 171 mM NaCl) supplemented with the appropriate selection antibiotic. At OD 600 = 0.5, cells were induced with 0.1 mM IPTG at 16°C for 16-18 h. Afterward, cells were spun down at 1,000 g for 10 min, and the pellet was resuspended in 3 ml NETN (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) for every 100 ml of culture. The suspension was sonicated for 3 × 10 s and cleared by centrifugation at 12,000 g for 10 min to produce the supernatant (crude extract) containing the GST fusion protein. To prepare GST fusion protein bound to glutathione agarose beads, 150 µl glutathione agarose slurry (sc-2009; Santa Cruz Biotechnology) was added for every 3 ml crude extract and mixed for 1 h at 4°C using a spiramix; beads were spun down at 1,000 g for 5 min and washed three times with 500 µl NETN, and an equal volume of NETN (150 µl) was added. GST proteins on glutathione agarose beads were stored at 4°C for up to 1 wk.

Cell culture and treatments
Human colon carcinoma BE cells (diploid cells that contain an oncogenic Kras-G13D mutation as well as the BRAF-G463V oncogenic mutation; a gift from Simon Wilkinson and Christopher Marshall, Institute of Cancer Research, London, UK; Petsalaki et al., 2018b), cervical carcinoma HeLa cells stably expressing LAP2b fused to RFP (a gift from Daniel Gerlich, Institute of Molecular Biotechnology, Vienna, Austria; Steigemann et al., 2009), or HeLa cells stably expressing α-tubulin fused to GFP (a gift from Jan-Michael Peters, Institute of Molecular Pathology, Vienna, Austria) were grown in DMEM (Gibco) containing 10% FBS at 37°C in 5% CO 2 . Cells were treated with 10 µM Chk2 inhibitor II (C3742; Millipore Sigma), 10 µM KU-55933 (sc-202963; Santa Cruz Biotechnology), 1 mM hydroxyurea (Sigma), 10 µM etoposide (Sigma), or 25 µM mirin (ab141182; Abcam) as appropriate. siRNA duplexes were transfected into BE cells 24 h before analysis using Lipofectamine 2000 (Invitrogen). For expression of GFP proteins, plasmids were transfected into cells in the absence or presence of appropriate siRNA duplexes 24 h before analysis or further drug treatment using Turbofect (Thermo Fisher Scientific). All cell lines used exhibited consistent morphology and growth properties and were negative for mycoplasma contamination.

Enrichment of cells in cytokinesis
BE cells were treated with 50 ng/ml nocodazole (Sigma) for 16 h, washed twice with prewarmed (37°C) PBS, and released in prewarmed fresh medium for 2 h before cell lysis or fixation. Microscopic examination had shown ∼30% of untransfected control cells were at midbody stage after this treatment.
Time-lapse imaging HeLa LAP2b:RFP or tubulin:GFP cells were seeded onto Petri dishes with a 30-mm glass base (Greiner), and an inverted fluorescence microscope (Observer D1; Zeiss) was used. Fluorescence or phase-contrast images were taken by using a 63× Plan Neofluor 0.75 NA Ph2 dry objective (Zeiss). Imaging was performed at 37°C in 5% CO 2 by using a Zeiss AxioCam MRm camera and Zeiss ZEN 2 acquisition software. Drugs were added to the medium immediately before filming as appropriate.

Indirect immunofluorescence microscopy and microscope image acquisition
For phospho-Aurora B-Ser331 or phospho-INCENP-Ser91 staining, cells were extracted in prewarmed (37°C) Phem buffer (60 mM Pipes, 25 mM Hepes, pH 7.0, 10 mM EGTA, and 4 mM MgSO 4 ) supplemented with 0.5% CHAPS and 100 nM microcystin (Sigma-Aldrich) for 5 min at room temperature, fixed with prewarmed (37°C) 4% paraformaldehyde in Phem buffer for 10 min at room temperature, permeabilized in 0.5% Triton X-100 in Phem buffer for 2 min at room temperature, washed twice with PBS, and immunostained. For phospho-ATM-1981 or total Chk2 staining, cells were fixed in ice-cold methanol for 5 min at −20°C, washed twice with PBS at room temperature, and immunostained. For all other fluorescence microscopy applications, cells were fixed in 4% paraformaldehyde in cytoskeleton buffer (1.1 M Na 2 HPO 4 , 0.4 M KH 2 PO 4 , 137 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM EGTA, 5 mM Pipes, and 5 mM glucose, pH 6.1) for 5 min at 37°C, permeabilized in 0.5% Triton X-100 in cytoskeleton buffer, washed twice with PBS at room temperature, and immunostained. For peptide competitions, 2 µg of the anti-pSer91 polyclonal antiserum was incubated with 100 µg peptide at 37°C for 1 h, and then used for immunostaining. FITC-or rhodamine-TRITC-conjugated (Jackson ImmunoResearch Laboratories) or Alexa Fluor 633-conjugated (Thermo Fisher Scientific) secondary antibodies were used as appropriate. DNA was stained with 10 µg/ ml DAPI (Biotium), and cells were mounted in Vectashield medium (Vector Laboratories). Images were collected by using a laser-scanning spectral confocal microscope (TCS SP8; Leica Microsystems), LASX software (Leica Microsystems), and a 63× Apochromat 1.40 NA oil objective. The low-fluorescence immersion oil (11513859; Leica Microsystems) was used, and imaging was performed at room temperature. Mean projections of image stacks were obtained by using the LASX software.
Multinucleate or midbody stage cells BE cells were fixed and stained with α-tubulin. Multinucleate cells or cells connected by midbodies were scored visually by immunofluorescence microscopy as described in the previous paragraph.

Broken bridges
Cells were fixed and stained for immunofluorescence microscopy as appropriate. The percentage of broken chromatin (DAPI) or LAP2b:RFP bridges was calculated as follows: (number of bridges that appear broken / total number of bridges) × 100%.

Quantification of fluorescence signals
Fluorescence intensity signals at midbodies were quantified using the LASX polygon tool by analyzing an image area of 2 µm 2 around each FB, and intensity values were normalized versus background values obtained by analyzing an identical area within the cell immediately adjacent on the midbody by subtracting the background-signal value from the midbody value (Petsalaki and Zachos, 2016;Waters, 2009). After subtraction of the background, the average values from control or mutant midbodies were calculated and were then all divided with the control midbodies-average value to obtain the relative midbody intensity values plotted (i.e., relative to control = 1), for comparison reasons.
To analyze midbody thickness, the diameter of each microtubule bundle at the midbody was measured using the LASX line tool and the average value calculated.
Actin-patch fluorescence intensity signals were quantified using the LCS Lite polygon tool by analyzing an image area of 40 µm 2 around the base of the DNA bridge, and intensity values were normalized versus background values obtained by analyzing an identical area within the cell near the base of the DNA bridge, by subtracting the background-signal value from the actin-patch value (Dandoulaki et al., 2018). After background subtraction, the average values from control or mutant actin patches were calculated and then all divided with the control actin patch average value to obtain the relative intensity values plotted (i.e., relative to control = 1).
For coimmunoprecipitation, 1 mg cell lysate was incubated with 1 µg antibody for 16 h followed by the addition of 10 µl protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) for 1 h at 4°C.
For GST pull-down, 1 mg cell lysate or 50 µl in vitro transcription-translation reactions (Fig. 4 H ;and Fig. 5,K and N) was incubated with 1 µg GST protein on glutathione-agarose beads (sc-2009; Santa Cruz Biotechnology) for 4 h at 4°C.
After incubation with the beads, samples were spun down and washed three times with immunoprecipitation kinase buffer, and precipitated proteins on beads were analyzed by SDS-PAGE, followed by Western blotting, autoradiography ( 32 P), or phosphorimaging ( 35 S).

In vitro transcription-translation and phosphorimager analysis
For in vitro transcription-translation, the TnT Quick Coupled Transcription/Translation System (Promega) was used. Briefly, 0.5 µg plasmid DNA template was incubated with 40 µl TnT T7 Quick Master Mix and 10 µCi 35 S-methionine per 50 µl reaction for 60 min at 30°C. Radiolabeled ( 35 S) samples transferred onto nitrocellulose membrane were analyzed by phosphorimaging using the Sapphire Biomolecular Imager (Azure Biosystems).
Growth curves BE cells were transfected with WT, Ser91A, or Ser91D pcDNA3.1/ V5-INCENP plasmids for 24 h. Cells were then replated in 30-mm dishes at 2 × 10 5 cells per dish for another 0-3 d and counted with a hemocytometer. Viable or dead cells were distinguished by trypan blue exclusion and optical microscopy.

Densitometry
Densitometric analysis of bands was performed using ImageJ (National Institutes of Health).

Statistical analysis and repetitions
For midbody fluorescence intensity, a minimum of 10 cells per experiment from two independent experiments were analyzed per treatment (n ≥ 20), and SD was calculated. For the frequency of cells at midbody stage or in prometaphase, SD from the mean from three independent experiments was calculated, and at least 300 cells per experiment and per treatment (n > 900) were analyzed. For growth curves, two replicates from three independent experiments (n = 6) were analyzed and the SD from the mean was calculated. For the frequency of broken chromatin bridges or γ-H2AX-positive or LAP2b:RFP bridges, at least 50 cells with bridges per experiment from three independent experiments were scored blindly, and the SD was calculated (n > 150). For the frequency of cells exhibiting Chmp4c:GFP-mislocalization ( Fig. S1 L), at least 30 cells per experiment from three independent experiments (n > 90) were analyzed and the SD was calculated. For actin-patches fluorescence (Fig. S4 H), at least 30 cells per experiment from three independent experiments were examined (n > 90), and the SD was calculated. Statistically significant differences among three or more groups were determined by one-way ANOVA followed by the Student's t test between two groups. No statistical method was used to predetermine sample size.
Immunoprecipitations (Figs. 3 B, 4 G, and 5 G) and GST pulldowns (Fig. 4 H;and Fig. 5, were repeated three times. GFP-Trap, kinase assays, and all other gels were done twice, and representative gels are shown. Online supplemental material Fig. S1 shows faster cleavage of the intercellular canal and mislocalized Chmp4c:GFP at the midbody in Chk2-deficient cells compared with controls, and localization of Aurora B or INCENP to early midbodies in normally segregating cells. Fig. S2 shows faster cleavage of the intercellular canal in cells expressing Ser91A-INCENP compared with controls, localization of phosphorylated Aurora B-Ser331 at the midbody in cells expressing GFP:INCENP(FB), and specificity of the anti-pChk2-T68, anti-pChk2-T68, or anti-pINCENP-Ser91 phospho-specific antibodies. Fig. S3 shows midbody indices in cells expressing Ser91D GFP: INCENP, localization of Mklp2 or Cep55 to early or late midbodies, binding of Aurora B, Survivin or Borealin with WT or mutant GFP:INCENP, and accelerated cleavage of the intercellular canal after ATM-inhibition in cytokinesis compared with control cells. Fig. S4 shows breakage of LAP2b:RFP bridges in cytokinesis, formation of actin patches, localization of WT, Ser91A, or Ser91D V5-INCENP, and localization of Rad50 to the midbody in cytokinesis with chromatin bridges. Fig. S5 shows ATM or INCENP localization in cells expressing ΔC20 GFP:Nbs1, and localization of Nbs1, phospho-Aurora B-Ser331, or V5-INCENP to the midbody in ATM-deficient or MRN-deficient cells in cytokinesis with chromatin bridges. Video 1 shows a control HeLa tubulin:GFP cell exhibiting midbody disassembly in cytokinesis. Video 2 shows a HeLa tubulin:GFP cell exhibiting midbody disassembly in the presence of Chk2 inhibitor II. Video 3 shows a control HeLa tubulin:GFP cell exhibiting cleavage of the intercellular canal in cytokinesis. Video 3 and Video 4 show HeLa tubulin:GFP cells exhibiting cleavage of the intercellular canal in the presence of, respectively, Chk2 inhibitor II or the ATM inhibitor KU-55933. Video 5 shows cleavage of the intercellular canal in ATM-deficient cells. Video 6 shows a control HeLa LAP2b:RFP cell exhibiting a stable LAP2b-positive intercellular canal in cytokinesis. Video 7 shows a HeLa LAP2b: RFP cell exhibiting breakage of the LAP2b-positive intercellular canal in the presence of Chk2 inhibitor II. Video 8 shows a HeLa LAP2b:RFP cell exhibiting breakage of the LAP2b:RFP bridge in the presence of Chk2 inhibitor II. Video 9 shows a HeLa LAP2b: RFP cell exhibiting breakage of the LAP2b-positive intercellular canal in the presence of KU-55933. Video 10 shows a HeLa LAP2b:RFP cell exhibiting breakage of the LAP2b:RFP bridge in the presence of KU-55933. Values represent mean ± SD from n cells. (P) Frequency of bi/multinucleate or prometaphase BE cells. Values represent mean ± SD from three independent experiments (n > 90). ***, P < 0.001 (ANOVA and Student's t test). S, serine. Scale bars, 5 µm.