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. 2018 Jul;559(7712):54-60.
doi: 10.1038/s41586-018-0242-8. Epub 2018 Jun 20.

Nuclear F-actin and Myosins Drive Relocalization of Heterochromatic Breaks

Affiliations
Free PMC article

Nuclear F-actin and Myosins Drive Relocalization of Heterochromatic Breaks

Christopher P Caridi et al. Nature. .
Free PMC article

Abstract

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

Conflict of interest statement

The authors declare no competing interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Actin nucleators mediate relocalization of heterochromatic DSBs
(a) IF and quantification of Kc cells fixed 60 min after IR show γH2Av foci in DAPI-bright (dashed circle) or total focus number, after treatment with LatB (+) or control (Ctrl, −). ****P<0.0001, ***P=0.0008 vs Ctrl, n>300 cells/treatment. (b) Wb or qPCR analyses show RNAi depletion efficiencies for indicated proteins in Kc cells. Actin or a background band (*) are used as loading controls. (c) Quantification of total γH2Av foci for Fig. 1a. (d) IF and quantification show γH2Av foci at the nuclear periphery (Lamin), or total focus number, 60 min after IR after indicated RNAi depletions in Kc cells. Only middle Z-stacks were used for quantifications. ****P<0.0001, n>200 cells/RNAi. (e) As in a except cells treated with CK666. ****P<0.0001, *P=0.00293, n>300 cells/treatment. (f–g) Quantification of γH2Av foci in DAPI-bright or total focus number, after 60 min treatment with and release from LatB or CK666. ****P<0.0001, n>200 cells per condition. (h) Quantification of Kc cells fixed 60 min after IR shows γH2Av foci in DAPI-bright, or total focus number, after RNAi depletions of actin nucleators Spire or the formin Dia. ****P<0.0001 vs Ctrl, n>300 cells/RNAi. (i) qPCR analysis shows Arp3 RNAi depletion efficiency in NIH3T3 cells. (j) Quantification of total γH2Av foci for Fig. 1b. (k) Quantification in NIH3T3 G2 cells treated with indicated chemicals, and fixed 60 min after IR, shows γH2AX foci in DAPI-bright or total focus number ****P<0.0001 vs Ctrl, n>37 cells/treatment. (l) Quantification of total γH2Av foci for Fig. 1c. Scale bars: 1 μm. Error bars: mean ± SEM in c, and ± SD of at least three independent experiments in a,d–h,k,l. P values calculated with two-tailed Mann-Whitney test.
Extended Data Figure 2
Extended Data Figure 2. Actin nucleators mediate relocalization of heterochromatic DSBs
(a) Images at 10 min after IR (+) and quantification before (−) and after IR of Mu2 and ATRIP foci in cells expressing GFP-Mu2 or GFP-ATRIP and mCh-HP1a, after indicated RNAi depletions. n>48 cells for ATRIP; n>21 cells for Mu2. (b) Quantification of Rad51 foci in cells fixed 60 min after IR and processed for IF with anti-Rad51 antibodies after indicated RNAi depletions, show the average number of foci relative to DAPI-bright or total foci. Smc5/6 RNAi results in abnormal formation of Rad51 foci in heterochromatin,,, and is used as positive control. ****P<0.0001 vs Ctrl, unpaired t-test with Welch’s correction, n>300 cells/RNAi. (c) Images and quantifications of Smc6 and Nse2 colocalizing with the mCh-HP1a domain before (−) IR and forming foci before and 15 min after (+) IR in cells expressing GFP-Smc6 or GFP-Nse2 and mCh-HP1a after indicated RNAi depletions. n=35 cells for Nse2 and n≥23 cells for Smc6. (d) Quantification at 30 min after IR of γH2Av foci colocalizing with FHA-tagged Arp2 or with Arpc3B after indicated RNAi depletions. ****P<0.0001, two-tailed Mann-Whitney test, n≥31 cells/RNAi. (e) IF of γH2Av foci colocalizing with Arpc3B and quantification of total focus number for Fig. 1f. (f) Quantification at indicated times after IR of the percentage of γH2Av foci colocalizing with FHA-tagged Arp2 relative to H3K9me3. n=30. (g) Quantification of total foci for Fig. 1d. (h) Western blot analyses show RNAi depletion efficiency for Arpc3B. Tubulin is used as loading control. (i) qPCR analysis shows Mre11 RNAi efficiency. Scale bars: 1 μm. Error bars: mean ± SEM in a,c,d,f, and ± SD of at least three independent experiments in b,g.
Extended Data Figure 3
Extended Data Figure 3. Nuclear F-actin is required for relocalization
(a) Western blot showing nuclear vs cytoplasmic actin levels in cells stably transfected with F-actCB-GFP-NLS. HP1a and Tubulin were used as loading controls and to check for purity of the fractions. Consistent with previous studies, expression of F-actCB-GFP-NLS does not alter nuclear actin levels, providing a non-invasive method for nuclear actin detection in vivo. (b) Frames of time-lapse experiments (Fig. 2a, Suppl. Videos 1,2) of cells expressing F-actCB-GFP-NLS and mCh-HP1a signals treated (+) or non-treated (−) with IR. Time points are from IR (+IR Video) or the start of imaging (-IR Video). (c) IF and quantification of cells stained with phalloidin show the formation of canonical nuclear actin filaments (highlighted in red) at indicated timepoints after IR. ****P<0.0001 for 10 min vs 0 min or 30 min, two-tailed Mann-Whitney test, n≥39 cells/timepoint. Error bars: SD of at least three independent experiments. (d) IF and staining for GFP, phalloidin, and γH2Av, of cells expressing F-actCB-GFP-NLS, show examples of actin filaments coated with damage foci in the absence (−) of triton extraction (TE), and actin puncta colocalizing with γH2Av foci after (+) TE. Zoomed details highlight colocalizations. (e) IF and staining for HA and phalloidin (Phall) of cells expressing FHA-Arp2 show enrichments of Arp2 along nuclear actin filaments, including at the base of actin branches (arrowheads). (f) Measurement of F-actin branch angles in response to IR, in time lapse experiments of cells expressing F-actCB-GFP-NLS. n=54 angles. (g) Quantification of the average duration of IR-induced actin filaments in time lapse experiments of cells expressing F-actCB-GFP-NLS. n=162 actin filaments. Dashed red vertical lines indicates median values in f,g. Scale bars: 1 μm.
Extended Data Figure 4
Extended Data Figure 4. Nuclear F-actin is required for relocalization
(a) Frames from time-lapse experiment show cells expressing F-actCB-GFP-NLS and Mu2-mCh (Fig. 2c, Suppl. Video 3) with an example of a Mu2 focus moving along an actin filament. Time indicates min from beginning of focus movement along the filament. Mu2 focus tracking (bottom panels) was done in Imaris. (b) Selected frames from cells expressing F-actCB-GFP-NLS and Mu2-mCh or HP1a-mCh show examples of filament directionality at indicated time points after IR, i.e., filaments emerging from the heterochromatin domain periphery (top), from repair foci (middle) or from the nuclear periphery (bottom). Percentages indicate the frequency at which each behavior is observed. n≥44 filaments. (c) Quantification of the length of actin filaments departing from the heterochromatin domain relative to the average distance between the HP1a domain periphery and the nuclear periphery. n>140 filaments. Dashed red vertical line: median value. (d) IF and staining for FLAG (actin-WT), phalloidin (Phall), and γH2Av, of cells expressing FLAG-actin-WT-NLS, show examples of actin signals in cells processed by triton extraction before fixation, 10 min after IR. Zoomed details highlight colocalizations. Scale bar: 1 μm. (e) IF of cells expressing FLAG+NLS-tagged versions of actin R62D and S14C as indicated shows colocalizations with γH2Av foci. Quantification shows total γH2Av foci for Fig. 2d. (f) Quantification of γH2Av foci colocalizing with FLAG-actin-WT-NLS 10 min after IR, or total number of foci, after indicated RNAi depletions. ****P<0.0001, n≥14 cells and n≥158 foci/RNAi. (g) qPCR analysis shows Ipo9 RNAi depletion efficiency. Ipo9 depletion specifically blocks the transfer of actin monomers to the nucleus,. (h) Quantification of total γH2Av foci for Fig. 2e. (i) Quantification of cells fixed 60 min after IR shows γH2Av foci at the nuclear periphery (lamin), or total focus number, after indicated RNAi depletions. Only the middle Z-stack was used for quantifications. ****P<0.0001, n≥215 cells/experiment. Error bars: SEM in e,h and SD of three independent experiments in f,i. P values calculated with two-tailed Mann-Whitney test.
Extended Data Figure 5
Extended Data Figure 5. Relocalization relies on nuclear myosins
(a) Western blot analysis of cells expressing FHA-tagged components as indicated, shows RNAi depletion efficiency for indicated proteins. (*) Background bands used as loading controls. (b) Quantification shows total γH2Av foci for Fig. 3a. (c) Quantification of cells fixed 60 min after IR shows γH2Av foci at the nuclear periphery (lamin), or total focus number, after indicated RNAi depletions. ****P<0.0001 vs Ctrl, Only the middle Z-stack was used for quantifications. Ctrl RNAi as in Extended Data Fig. 1d. (d) Quantification of cells fixed 60 min after IR shows γH2Av foci in DAPI-bright, or total focus number, after indicated RNAi depletions. ****P<0.0001, ***P=0.003, **P≤0.0022, *P=0.0402 vs Ctrl, n>200 cells/RNAi. (e) Quantification of total γH2Av foci for Fig. 3b. (f) Images at 10 min after IR and quantification at 0 and 10 min after IR of Mu2, and ATRIP foci in cells expressing GFP-Mu2 or GFP-ATRIP and mCh-HP1a, after indicated RNAi depletions. n≥75 cells for ATRIP and n≥17 cells for Mu2. (g) Images and quantifications of Smc6 and Nse2 colocalizing with the mCh-HP1a domain before (−) IR and forming foci at 0 and 15 min after (+) IR in cells expressing GFP-Smc6 or GFP-Nse2 and mCh-HP1a after indicated RNAi depletions. n≥31 cells for Nse2 and n≥21 cells for Smc6. In f,g, Myo indicates RNAi depletion of Myo1A+Myo1B+MyoV. Scale bars: 1 μm. Error bars: mean ± SEM in b,f,g and ± SD of at least three independent experiments in c,d,e. All P values calculated with two-tailed Mann-Whitney test except one-tailed Mann-Whitney test was used for Myo1B RNAi in d.
Extended Data Figure 6
Extended Data Figure 6. Relocalization relies on nuclear myosins
(a) Quantification of Rad51 foci in cells fixed 60 min after IR after indicated RNAi depletions show the average number of foci relative to DAPI-bright or total foci. ****P=0.0001 vs Ctrl, n>300 cells/RNAi. Ctrl and Smc5/6 RNAi as in Extended Data Fig. 2b. (b) Western blot validation of the antibodies used in c and in Fig. 3c shows loss of Rad50, TopBP1 and dPIAS bands after corresponding RNAi depletions. (c) Wb analysis of the same Ip shown in Fig. 3c probed with antibodies for dPIAS or TopBP1, with actin as the loading control. (d) Quantification at 30 min after IR of γH2Av foci colocalizing with FHA-Myo1A, GFP-Myo1B, MyoV, or Unc45 after indicated RNAi depletions. ****P<0.0001, n>21/cells/RNAi. (e) IF at 10 min after IR and quantification at indicated time points after IR of γH2Av foci colocalizing with FHA-Myo1A, GFP-Myo1B, MyoV, or Unc45. (f) Quantification at indicated times after IR of the percentage of γH2Av+FHA-Myo1A, GFP-Myo1B, MyoV, or Unc45, relative to H3K9me3. n=30 cells/experiment/timepoint. (g) Quantification of Kc cells fixed 60 min after IR shows γH2Av foci in DAPI-bright, or total focus number, after treatment with BDM, MyoVin or controls. ****P<0.0001, **P<0.008, n≥300 cells/treatment. (h) Quantification of γH2Av foci in DAPI-bright or total foci, after 60 min treatment with and release from BDM or MyoVin. ****P<0.0001, n≥200 cells/treatment. (i) As in g, except NIH3T3 cells were used and γH2AX foci were quantified. Ctrl RNAi as Extended Data Fig. 1k. ****P<0.0001, n≥37 cells and n≥2524 foci/treatment. Error bars: mean ± SEM in d–f and ± SD of at least three independent experiments in a,g–i. All P values calculated with two-tailed Mann-Whitney test.
Extended Data Figure 7
Extended Data Figure 7. Actin nucleators and myosin drive directed motions of heterochromatic DSBs
(a) Selected images and quantification at indicated time points of Mu2 foci in GFP-Mdc1/Mu2 and mCh-HP1a expressing cells show no effect of the imaging conditions used in Fig. 4 on the ability of cells to divide or the total number of repair foci. Only representative time points are shown. n=82 cells. Scale bar: 1 μm. (b) Left and middle: Quantification and violin plot display of the frequency of heterochromatic (HC) Mu2/Mdc1 foci moving relative to the HP1a domain during the time lapse experiments shown in Fig. 5a,b, after indicated RNAi depletions. White box: median. Red line: mean. Vertical black lines: values that occur 95% and 50% of the time. ‘Dynamic HC foci’ include foci moving from inside the HP1a domain to: the nuclear periphery (imNP); outside the HP1a domain (imo); or the periphery of the HP1a domain (imp). ‘Static HC foci’ include foci remaining inside (isi) or at the periphery of the HP1a domain (psp) throughout the 1 h timecourse. Colors reflect the categories analyzed in Fig. 5b. Right: Quantification of the average number of Mu2 foci from Fig. 5a,b and b, after indicated RNAi depletions. *P<0.05,**P<0.006, ***P=0.0003; ****P<0.0001, two-tailed Mann-Whitney test, n≥217 foci/RNAi. (c) Quantification of the percentage of heterochromatic (HC) and euchromatic (EU) Mu2/Mdc1 foci moving to the nuclear periphery in time lapse experiments from Fig. 5a,b. ****P<0.0003; two-tailed Mann-Whitney test, n=162 foci. (d) LDM analysis of Mu2/Mdc1 foci that reach the nuclear periphery from Fig. 4b. Each colored horizontal bar represents a focus and its duration in each nuclear compartment, as indicated. The black segments under each bar are LDMs for each focus. (e) MSD curves generated from the positional data corresponding to the time points that contain LDMs in d. Longer (left) and shorter (right) LDMs are presented as independent graphs for clarity. (f) Quantification of the analysis shown in Fig. 4d and d shows the average duration of directed motions (LDMs) or non-directed motions in the nuclear positions defined in d. P<0.0001 for directed motions in 3 vs 1,2,4, unpaired t-test. (g) Quantification of LDM durations for euchromatic foci shown in Fig. 4d, after indicated RNAi depletions. (h) Quantification and violin plot of Mu2 focus clustering events in time-lapse experiments for Fig. 4d. Graphical display as in b. An example of clustering is highlighted in Suppl. Video 4. ***P=0.0003, two-tailed Mann-Whitney test, n≥20 cells/RNAi. Error bars: SEM.
Extended Data Figure 8
Extended Data Figure 8. Actin nucleators and myosins promote heterochromatin repair and stability
(a) Wb and qPCR analysis show RNAi depletion efficiency for Rad21 and Slmb as indicated. Tubulin is a loading control for Wb. (b) FISH analysis and quantification show the effect of indicated RNAi on the number of cells with ≥3 AACAC or 359bp satellites, reflecting disruption of homologous and/or sister pairing. **P=0.0472; ****P<0.0001 two-tailed Mann-Whitney test, n>120 cells/RNAi. (c) Quantification shows γH2Av foci associated (+) or non-associated (−) with H3K9me2 signals 20 h after IR following indicated RNAi depletions. **P<0.02; ****P<0.0001 two-tailed Mann-Whitney test, n>400 cells/RNAi. (d) Quantification of Kc cells from the experiment in Fig. 5a shows γH2Av foci non-associated (−) with H3K9me2 signals 20 h after IR and following indicated RNAi depletions. (e) Quantification of NIH3T3 cells from the experiment in Fig. 5b shows γH2AX foci non-associated (−) with DAPI-bright signals 16 and 24 h after IR and following indicated RNAi depletions. (f) Quantification of micronuclei in non-irradiated Kc cells from Fig. 4d. (g) Quantification of micronuclei in non-irradiated NIH3T3 cells from Fig. 4e. (h) qPCR and Wb analyses show RNAi depletion efficiency of indicated components in 3rd instar larvae for i and Fig. 5f. (i) Images and quantification of chromosome aberrations in karyotypes from Fig. 5f. Images show extra satellites (arrows), chromosome fusions (arrowhead), chromosome arm losses (dashed circles). The diagram of Drosophila chromosomes indicates the position of the main satellites detected by FISH. Aneuploidies and chromosome fusions from Fig. 5f were further categorized to highlight rearrangements involving centromeric regions or chromosomes that are predominantly heterochromatic (4th or Y). Error bars: mean ± SEM in a(RNAi),b,f and ± SD of at least three independent experiments in a(actin),c–e.
Figure 1
Figure 1. Actin nucleators mediate relocalization of heterochromatic DSBs
(a) Immunofluorescence (IF) and quantification of Kc cells fixed at indicated timepoints after IR show γH2Av foci in DAPI-bright following indicated RNAi. ****P≤0.0001, n≥100 cells/RNAi/timepoint. (b) IF and quantification of NIH3T3 G2 cells at indicated timepoints after IR show γH2AX foci associated with DAPI-bright chromocenters following indicated RNAi. ****P<0.0001, ***P=0.0004 vs Ctrl, n≥20 cells/RNAi/timepoint. (c) As in a., IF performed 60 min after IR. ****P<0.0001 and ***P=0.0009 vs Ctrl, **P=0.0019, n>250 cells/RNAi/timepoint. (d) As in a. ****P<0.0001 vs Ctrl, n>250 cells/RNAi. (e) Immunoprecipitation (Ip) of FHA-Nse2 or GFP-Arp3 in Kc cells before (−) or after (+) IR and Western blot (Wb) analysis with indicated antibodies. Actin, background band (*): loading controls. WCE: whole cell extract. (f) IF and quantification in DAPI-bright at indicated timepoints after IR of γH2Av foci colocalizing with FHA(FLAG-HA)-Arp2 or Arpc3B. Arrows: colocalizations in DAPI-bright. n>30 Kc cells/timepoint/experiment. (g) As in f, colocalizations between γH2Av and Arp2 10 min after IR, following indicated RNAi. ****P<0.0001, n>22 cells/RNAi/experiment. Scale bars: 1 μm. Error bars: mean ± SEM in a,b,f,g and ± SD of at least three independent experiments in c,d. Grey headings above each graph indicate the compartments to which quantifications refer. P values calculated with two-tailed Mann-Whitney test.
Figure 2
Figure 2. Nuclear F-actin is required for relocalization
(a) Frames of Suppl. Video 2 and quantification of Kc cells expressing F-actCB-GFP-NLS and mCherry(mCh)-HP1a (heterochromatin mark) show dynamic filaments in cells treated (+) or non-treated (−) with IR. Arrowheads: filaments reaching the nuclear periphery. Timepoints are after IR. P<0.0001, + vs − IR; n>22 cells/experiment. (b) As in a except cells treated with indicated RNAi or CK666. P<0.0001, Ctrl vs Arp2/3 RNAi, DMSO vs CK666, n≥19 cells/treatment/experiment. (c) Frames of Suppl. Video 3 show Mu2 focus moving along actin filaments. Timepoints relative to start of focus movement. (d) IF of γH2Av foci colocalizing with FLAG-NLS-actin WT, R62D, or S14C at indicated timepoints. ****P<0.0001, ***P=0.0006 n≥40 cells/timepoint/experiment. (e) Quantification of γH2Av foci in DAPI-bright at indicated time points, in cells expressing indicated actin forms after Ipo9 RNAi. ***P=0.0005, **P=0.0019, n≥47 cells/timepoint/experiment. Scale bars: 1 μm. Error bars: mean ± SEM. P values calculated with extra sum-of-squares F-test, nonlinear regression for curve fitting in a,b and two-tailed Mann-Whitney test in d,e.
Figure 3
Figure 3. Relocalization relies on nuclear myosins
(a) Quantification of Kc cells fixed at indicated timepoints shows γH2Av foci in DAPI-bright following indicated RNAi or expression of MyoV forms. ****P<0.0001, ***P=0.0004, **P≤0.0033, n≥99 cells/RNAi or cell line/timepoint. (b) As in a, 1 h after IR. ****P<0.0001, n≥227 cells/RNAi. Ctrl and Smc5/6 as in Fig. 1d. (c) Ip of GFP-Myo1A, GFP-Myo1B or FHA-MyoV with Smc5/6 before (−) and after (+) IR and Wb analysis with indicated antibodies. (d) IF and quantification in DAPI-bright of γH2Av foci colocalizing with FHA-Myo1A, GFP-Myo1B, MyoV, or Unc45, at indicated timepoints. n ≥30 cells/timepoint/experiment. Scale bars: 1 μm. (g) As in d, 10 min after IR, after indicated RNAi. ****P<0.0001 vs Ctrl, n >24 cells/experiment. Error bars: mean ± SEM in a,d,e and ± SD of at least three independent experiments in b.
Figure 4
Figure 4. Actin nucleators and myosins drive directed motions of heterochromatic DSBs
(a) MSD analysis of heterochromatic (HC) or euchromatic (EU) GFP-Mu2/Mdc1 foci (i.e., formed inside or outside the HP1a domain, respectively) after IR, or GFP-CenpA foci before (−) and after (+) IR. P<0.0001 for all comparisons, n>19 tracks/category. (b) MSD analysis of HC Mu2 foci from a that: remain inside HP1a domain; move to domain periphery; remain at domain periphery; or move to nuclear periphery. P<0.0001 for all comparisons vs ‘to nuclear periphery’ category. n=41 foci (c) LDM analysis of foci that reach nuclear periphery in b. Duration and length of LDMs (black lines) are adjusted relative to a ‘pseudo-trajectory’ defined by average time foci are in each nuclear space. n=28 foci. (d) 3D reconstructions in Imaris and MSD analyses of GFP-Mu2/Mdc1 foci that leave the heterochromatin domain (mCh-HP1a) and reach the nuclear periphery, or euchromatic foci, after indicated RNAi depletions. P<0.0001 vs Ctrl, n≥28 HC and n≥19 EU foci/RNAi. (e) Quantification of LDM durations of all heterochromatic foci in d. ****P<0.0001 vs Ctrl, n≥30 cells/RNAi. Scale bars: 1 μm. Error bars: mean ± SEM in a–e. P values calculated with extra sum-of-squares F-test, nonlinear regression for curve fitting for a,b,d and two-tailed Mann-Whitney test in e.
Figure 5
Figure 5. Nuclear F-Actin and myosins promote heterochromatin integrity
(a) IF and quantification of Kc cells fixed 20 h after IR show γH2Av foci associated (+) with H3K9me2 after indicated RNAi or expression of actin forms. ****P<0.0001, n>65 cells/RNAi and n>220 cells/actin-expressing line. (b) As in a except NIH3T3 G2 cells stained for γH2AX and PH3. ****P<0.0001, ***P=0.0008, n ≥20 cells/RNAi. (c) IR sensitivity of Kc cells after indicated RNAi or expression of actin forms. Rad51 RNAi: positive control. P<0.0001 vs Ctrl RNAi or Act-WT, n>1324 cells/RNAi/dose. (d) IF and quantification of micronuclei (arrow) in cells stained for H3K9me2 and nuclear periphery (Nup62), after indicated RNAi or expression of actin forms and IR. ***P<0.0009, n>311 cells/RNAi; ****P<0.0001, n >340 cells/actin expressing line. +/−: micronuclei with/without H3K9me2 signals. (e) As in d, except NIH3T3 cells stained for H3K9me3. ****P<0.0001 vs Ctrl; n>1450 cells/RNAi. (f) FISH and quantification of larval karyotypes stained for indicated satellites show chromosome abnormalities in mutants or RNAi depleted flies vs controls w1118 or ActGAL4/+. Arrows: chromosome fusions. Dashed circle: arm loss. ****P<0.0001, n>36 karyotypes/genotype. Scale bars: 1 μm in a,d; 5 μm in b,e,f. Error bars: mean ± SEM in a(RNAi),b,f or ± SD of at least three independent experiments in a(actin),c–e. P values calculated with two-tailed Mann-Whitney test in a,b,d(RNAi); unpaired t-test with Welch’s correction in d(actin),e,f; extra sum-of-squares F-test, nonlinear regression for curve-fitting in c. (g) Heterochromatin relocalization pathway model: DSBs are resected inside the heterochromatin domain; Mre11 and HP1a promote the loading of Arp2/3 and nuclear myosins to DSBs; Arp2/3 activation by Scar and Wash induce formation of actin filaments reaching nuclear periphery; Unc45 recruitment by Smc5/6 activates nuclear myosins to ‘walk’ along filaments, thus relocalizing DSBs to nuclear pores or INMPs,,; HR continues while filaments disassemble. Relocalization prevents ectopic recombination by isolating damaged sites and their homologous templates (grey lines) from undamaged heterochromatic repeats before strand invasion.

Comment in

  • Breakdancing on actin.
    Strzyz P. Strzyz P. Nat Rev Mol Cell Biol. 2018 Aug;19(8):485. doi: 10.1038/s41580-018-0036-8. Nat Rev Mol Cell Biol. 2018. PMID: 29967438 No abstract available.
  • Breakdancing on actin.
    Strzyz P. Strzyz P. Nat Rev Genet. 2018 Aug;19(8):469. doi: 10.1038/s41576-018-0033-y. Nat Rev Genet. 2018. PMID: 29967492 No abstract available.

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