Skip to main content
SpringerLink
Log in

Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells

  • Protocol
  • First Online:
Homologous Recombination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2153))

Abstract

Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

Colby See and Deepak Arya have contributed equally for this manuscript.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Peng JC, Karpen GH (2008) Epigenetic regulation of heterochromatic DNA stability. Curr Opin Genet Dev 18(2):204–211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chiolo I, Tang J, Georgescu W et al (2013) Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin. Mutat Res 750(1–2):56–66

    Article  CAS  PubMed  Google Scholar 

  3. Amaral N, Ryu T, Li X et al (2017) Nuclear dynamics of heterochromatin repair. Trends Genet 33(2):86–100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Caridi PC, Delabaere L, Zapotoczny G et al (2017) And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break. Philos Trans R Soc Lond Ser B Biol Sci 372(1731):pii: 20160291

    Article  Google Scholar 

  5. Hoskins RA, Carlson JW, Kennedy C et al (2007) Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science 316(5831):1625–1628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ho JW, Jung YL, Liu T et al (2014) Comparative analysis of metazoan chromatin organization. Nature 512(7515):449–452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hoskins RA, Carlson JW, Wan KH et al (2015) The release 6 reference sequence of the Drosophila melanogaster genome. Genome Res 25(3):445–458

    Article  PubMed  PubMed Central  Google Scholar 

  8. James TC, Eissenberg JC, Craig C et al (1989) Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur J Cell Biol 50(1):170–180

    CAS  PubMed  Google Scholar 

  9. Riddle NC, Minoda A, Kharchenko PV et al (2011) Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin. Genome Res 21(2):147–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lachner M, O’Carroll D, Rea S et al (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410(6824):116–120

    Article  CAS  PubMed  Google Scholar 

  11. Dialynas GK, Terjung S, Brown JP et al (2007) Plasticity of HP1 proteins in mammalian cells. J Cell Sci 120(19):3415–3424

    Google Scholar 

  12. Horz W, Altenburger W (1981) Nucleotide sequence of mouse satellite DNA. Nucleic Acids Res 9(3):683–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ostromyshenskii DI, Chernyaeva EN, Kuznetsova IS et al (2018) Mouse chromocenters DNA content: sequencing and in silico analysis. BMC Genomics 19(1):151

    Article  PubMed  PubMed Central  Google Scholar 

  14. Rawal C, Caridi PC, Chiolo I (2019) Actin’ between phase separated domains for heterochromatin repair. DNA Repair. 81:102646

    Google Scholar 

  15. Kowalczykowski SC (2015) An overview of the molecular mechanisms of recombinational DNA repair. Cold Spring Harb Perspect Biol 7(11):pii: a016410

    Article  Google Scholar 

  16. Peng JC, Karpen GH (2007) H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 9(1):25–35

    Article  CAS  PubMed  Google Scholar 

  17. Peng JC, Karpen GH (2009) Heterochromatic genome stability requires regulators of histone H3 K9 methylation. PLoS Genet 5(3):e1000435

    Article  PubMed  PubMed Central  Google Scholar 

  18. Ryu T, Spatola B, Delabaere L et al (2015) Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nat Cell Biol 17(11):1401–1411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ryu T, Bonner MR, Chiolo I (2016) Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus 7(5):485–497

    Article  PubMed  PubMed Central  Google Scholar 

  20. Caridi CP, D’Agostino C, Ryu T et al (2018) Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559(7712):54–60

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dialynas G, Delabaere L, Chiolo I (2019) Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes. Exp Biol Med. 244(15):1362–1371

    Google Scholar 

  22. Beucher A, Birraux J, Tchouandong L et al (2009) ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J 28(21):3413–3427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chiolo I, Minoda A, Colmenares SU et al (2011) Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144(5):732–744

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kakarougkas A, Ismail A, Klement K et al (2013) Opposing roles for 53BP1 during homologous recombination. Nucleic Acids Res 41(21):9719–9731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Janssen A, Breuer GA, Brinkman EK et al (2016) A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes Dev 30(14):1645–1657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tsouroula K, Furst A, Rogier M et al (2016) Temporal and spatial uncoupling of DNA double strand break repair pathways within mammalian heterochromatin. Mol Cell 63(2):293–305

    Article  CAS  PubMed  Google Scholar 

  27. Delabaere L, Chiolo I (2016) ReiNF4rcing repair pathway choice during cell cycle. Cell Cycle 15(9):1182–1183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Colmenares SU, Swenson JM, Langley SA et al (2017) Drosophila histone demethylase KDM4A has enzymatic and non-enzymatic roles in controlling heterochromatin integrity. Dev Cell 42(2):156–169 e5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Janssen A, Colmenares SU, Lee T et al (2019) Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A. Genes Dev 33(1–2):103–115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Caridi CP, Plessner M, Grosse R et al (2019) Nuclear actin filaments in DNA repair dynamics. Nat Cell Biol 21(9):1068–1077

    Article  CAS  PubMed  Google Scholar 

  31. Guenatri M, Bailly D, Maison C et al (2004) Mouse centric and pericentric satellite repeats form distinct functional heterochromatin. J Cell Biol 166(4):493–505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li Q, Tjong H, Li X et al (2017) The three-dimensional genome organization of Drosophila melanogaster through data integration. Genome Biol 18(1):145

    Article  PubMed  PubMed Central  Google Scholar 

  33. Jakob B, Splinter J, Conrad S et al (2011) DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin. Nucleic Acids Res 39(15):6489–6499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Haaf T, Golub EI, Reddy G et al (1995) Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92(6):2298–2302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Maser RS, Monsen KJ, Nelms BE et al (1997) hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol Cell Biol 17(10):6087–6096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Scully R, Chen J, Ochs RL et al (1997) Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90(3):425–435

    Article  CAS  PubMed  Google Scholar 

  37. Liu Y, Li M, Lee EY et al (1999) Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage. Curr Biol 9(17):975–978

    Article  CAS  PubMed  Google Scholar 

  38. Lisby M, Barlow JH, Burgess RC et al (2004) Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118(6):699–713

    Article  CAS  PubMed  Google Scholar 

  39. Lisby M, Rothstein R (2004) DNA damage checkpoint and repair centers. Curr Opin Cell Biol 16(3):328–334

    Article  CAS  PubMed  Google Scholar 

  40. Costes SV, Chiolo I, Pluth JM et al (2010) Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization. Mutat Res 704(1–3):78–87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Delabaere L, Ertl HA, Massey DJ et al (2016) Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells. Aging Cell. 16(2):320–328

    Google Scholar 

  42. Caridi CP, Delabaere L, Tjong H et al (2018) Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells. Methods Enzymol 601:359–389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Stucki M, Clapperton JA, Mohammad D et al (2005) MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123(7):1213–1226

    Article  CAS  PubMed  Google Scholar 

  44. Dronamraju R, Mason JM (2009) Recognition of double strand breaks by a mutator protein (MU2) in Drosophila melanogaster. PLoS Genet 5(5):e1000473

    Article  PubMed  PubMed Central  Google Scholar 

  45. Madigan JP, Chotkowski HL, Glaser RL (2002) DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis. Nucleic Acids Res 30(17):3698–3705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Fernandez-Capetillo O, Lee A, Nussenzweig M et al (2004) H2AX: the histone guardian of the genome. DNA Repair 3(8–9):959–967

    Article  CAS  PubMed  Google Scholar 

  47. Wang B, Matsuoka S, Carpenter PB et al (2002) 53BP1, a mediator of the DNA damage checkpoint. Science 298(5597):1435–1438

    Article  CAS  PubMed  Google Scholar 

  48. Goldberg M, Stucki M, Falck J et al (2003) MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421(6926):952–956

    Article  CAS  PubMed  Google Scholar 

  49. Lou Z, Minter-Dykhouse K, Franco S et al (2006) MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol Cell 21(2):187–200

    Article  CAS  PubMed  Google Scholar 

  50. Chapman JR, Jackson SP (2008) Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage. EMBO Rep 9(8):795–801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Belin BJ, Cimini BA, Blackburn EH et al (2013) Visualization of actin filaments and monomers in somatic cell nuclei. Mol Biol Cell 24(7):982–994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Melak M, Plessner M, Grosse R (2017) Actin visualization at a glance. J Cell Sci 130(3):525–530

    PubMed  Google Scholar 

  53. Baarlink C, Wang H, Grosse R (2013) Nuclear actin network assembly by formins regulates the SRF coactivator MAL. Science 340(6134):864–867

    Article  CAS  PubMed  Google Scholar 

  54. Belin BJ, Lee T, Mullins RD (2015) DNA damage induces nuclear actin filament assembly by formin-2 and spire-(1/2) that promotes efficient DNA repair. Elife 4:e07735

    Article  PubMed  PubMed Central  Google Scholar 

  55. Plessner M, Melak M, Chinchilla P et al (2015) Nuclear F-actin formation and reorganization upon cell spreading. J Biol Chem 290(18):11209–11216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Iwaki T, Figuera M, Ploplis VA et al (2003) Rapid selection of Drosophila S2 cells with the puromycin resistance gene. Biotechniques 35(3):482–486

    Article  CAS  PubMed  Google Scholar 

  57. Ozaki T, Nagase T, Ichimiya S et al (2000) NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain. DNA Cell Biol 19(8):475–485

    Article  CAS  PubMed  Google Scholar 

  58. Galanty Y, Belotserkovskaya R, Coates J et al (2009) Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462(7275):935–939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Schmiedeberg L, Weisshart K, Diekmann S et al (2004) High- and low-mobility populations of HP1 in heterochromatin of mammalian cells. Mol Biol Cell 15(6):2819–2833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Riedl J, Crevenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5(7):605–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Johnson HW, Schell MJ (2009) Neuronal IP3 3-kinase is an F-actin-bundling protein: role in dendritic targeting and regulation of spine morphology. Mol Biol Cell 20(24):5166–5180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Burkel BM, von Dassow G, Bement WM (2007) Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell Motil Cytoskeleton 64(11):822–832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Goodarzi AA, Noon AT, Deckbar D et al (2008) ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol Cell 31(2):167–177

    Article  CAS  PubMed  Google Scholar 

  64. Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399

    Article  CAS  PubMed  Google Scholar 

  65. Spichal M, Fabre E (2017) The emerging role of the cytoskeleton in chromosome dynamics. Front Genet 8:60

    Article  PubMed  PubMed Central  Google Scholar 

  66. Lamm N, Masamsetti VP, Read MN et al (2018) ATR and mTOR regulate F-actin to alter nuclear architecture and repair replication stress. bioRxiv. https://doi.org/10.1101/451708

  67. Oshidari R, Strecker J, Chung DKC et al (2018) Nuclear microtubule filaments mediate non-linear directional motion of chromatin and promote DNA repair. Nat Commun 9(1):2567

    Article  PubMed  PubMed Central  Google Scholar 

  68. Cherbas L, Gong L (2014) Cell lines. Methods 68(1):74–81

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Ayoub N, Jeyasekharan AD, Bernal JA et al (2008) HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response. Nature 453(7195):682–686

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by NIH R01GM117376 and NSF Career 1751197 to I.C., and NIH T32 GM118289 to C.S. We would like to thank S. Keagy for insightful comments on the chapter, S. Jackson, and P. Hemmerich for plasmids, the Longo lab for NIH3T3 cells, and C. Caridi for his help with MSD and LDM scripts.

Author contributions: C.S. contributed to optimizing imaging approaches and analysis methods in Drosophila cells, and D.A. in mouse cells. D.A., C.S., and I.C. wrote the manuscript. E.L. generated data for Fig. 2. *C.S. and D.A. contributed equally to this manuscript.

Author information

Authors and Affiliations

  1. Molecular and Computational Biology Department, University of Southern California, Los Angeles, CA, USA

    Colby See, Deepak Arya, Emily Lin & Irene Chiolo

Authors
  1. Colby See
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deepak Arya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emily Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Irene Chiolo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Irene Chiolo .

Editor information

Editors and Affiliations

  1. Centro Andaluz de Biologı´a Molecular, y Medicina Regenerativa, CABIMER, Universidad de Sevilla, Sevilla, Spain

    Andrés Aguilera

  2. Institut Curie, PSL Research University, CNRS, UMR3348, F-91405, Orsay, France

    Aura Carreira

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

See, C., Arya, D., Lin, E., Chiolo, I. (2021). Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_32

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0644-5_32

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0643-8

  • Online ISBN: 978-1-0716-0644-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation