ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly

Abstract

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

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References

  1. 1.

    Poon IKH, Lucas CD, Rossi AG, Ravichandran KS. Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol. 2014;14:166–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Hochreiter-Hufford A, Ravichandran KS. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb Perspect Biol. 2013;5:a008748.

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Nagata S. Apoptosis and clearance of apoptotic cells. Annu Rev Immunol. 2018;36:489–517.

    CAS  PubMed  Google Scholar 

  4. 4.

    Atkin-Smith GK, Poon IKH. Disassembly of the dying: mechanisms and functions. Trends Cell Biol. 2017;27:151–62.

    CAS  PubMed  Google Scholar 

  5. 5.

    Shao W-H, Cohen PL. Disturbances of apoptotic cell clearance in systemic lupus erythematosus. Arthritis Res Ther. 2011;13:202.

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Baumann I, Kolowos W, Voll RE, Manger B, Gaipl U, Neuhuber WL, et al. Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus. Arthritis Rheum. 2002;46:191–201.

    PubMed  Google Scholar 

  7. 7.

    Gaipl US, Franz S, Voll RE, Sheriff A, Kalden JR, Herrmann M. Defects in the disposal of dying cells lead to autoimmunity. Curr Rheuma Rep. 2004;6:401–7.

    Google Scholar 

  8. 8.

    Lee CS, Penberthy KK, Wheeler KM, Juncadella IJ, Vandenabeele P, Lysiak JJ, et al. Boosting apoptotic cell clearance by colonic epithelial cells attenuates inflammation in vivo. Immunity. 2016;44:807–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Ait-Oufella H, Pouresmail V, Simon T, Blanc-Brude O, Kinugawa K, Merval R, et al. Defective mer receptor tyrosine kinase signaling in bone marrow cells promotes apoptotic cell accumulation and accelerates atherosclerosis. Arterioscler Thromb Vasc Biol. 2008;28:1429–31.

    CAS  PubMed  Google Scholar 

  10. 10.

    Atkin-Smith GK, Tixeira R, Paone S, Mathivanan S, Collins C, Liem M, et al. A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nat Commun. 2015;6:7439.

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Poon IKH, Chiu Y-H, Armstrong AJ, Kinchen JM, Juncadella IJ, Bayliss DA, et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature. 2014;507:329–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Tixeira R, Caruso S, Paone S, Baxter AA, Atkin-Smith GK, Hulett MD, et al. Defining the morphologic features and products of cell disassembly during apoptosis. Apoptosis. 2017;22:475–7.

    PubMed  Google Scholar 

  13. 13.

    Witasp E, Uthaisang W, Elenström-Magnusson C, Hanayama R, Tanaka M, Nagata S, et al. Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells. Cell Death Differ. 2007;14:1063.

    CAS  PubMed  Google Scholar 

  14. 14.

    Orlando KA, Stone NL, Pittman RN. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Exp Cell Res. 2006;312:5–15.

    CAS  PubMed  Google Scholar 

  15. 15.

    Fransen JH, Hilbrands LB, Ruben J, Stoffels M, Adema GJ, van der Vlag J, et al. Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin-17. Arthritis Rheum. 2009;60:2304–13.

    CAS  PubMed  Google Scholar 

  16. 16.

    Fransen JH, Hilbrands LB, Jacobs CW, Adema GJ, Berden JH, Van Der Vlag J. Both early and late apoptotic blebs are taken up by DC and induce IL-6 production. Autoimmunity. 2009;42:325–7.

    CAS  PubMed  Google Scholar 

  17. 17.

    Wickman GR, Julian L, Mardilovich K, Schumacher S, Munro J, Rath N, et al. Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs. Cell Death Differ. 2013;20:1293–305.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Fournel S, Neichel S, Dali H, Farci S, Maillère B, Briand J-P, et al. CD4+T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3. J Immunol. 2003;171:636–44.

    CAS  PubMed  Google Scholar 

  19. 19.

    Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol. 2001;3:339–45.

    CAS  PubMed  Google Scholar 

  20. 20.

    Sebbagh M, Renvoizé C, Hamelin J, Riché N, Bertoglio J, Bréard J. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol. 2001;3:346.

    CAS  PubMed  Google Scholar 

  21. 21.

    Lee N, MacDonald H, Reinhard C, Halenbeck R, Roulston A, Shi T, et al. Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc Natl Acad Sci USA. 1997;94:13642–7.

    CAS  PubMed  Google Scholar 

  22. 22.

    Rudel T, Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science. 1997;276:1571–4.

    CAS  PubMed  Google Scholar 

  23. 23.

    Tomiyoshi G, Horita Y, Nishita M, Ohashi K, Mizuno K. Caspase-mediated cleavage and activation of LIM-kinase 1 and its role in apoptotic membrane blebbing. Genes Cells. 2004;9:591–600.

    CAS  PubMed  Google Scholar 

  24. 24.

    Jiang L, Tixeira R, Caruso S, Atkin-Smith GK, Baxter AA, Paone S, et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat Protoc. 2016;11:655–63.

    CAS  PubMed  Google Scholar 

  25. 25.

    Doe C, Bentley R, Behm DJ, Lafferty R, Stavenger R, Jung D, et al. Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities. J Pharm Exp Ther. 2007;320:89–98.

    CAS  Google Scholar 

  26. 26.

    Koval AB, Wuest WM. An optimized synthesis of the potent and selective Pak1 inhibitor FRAX-1036. Tetrahedron Lett. 2016;57:449–51.

    CAS  Google Scholar 

  27. 27.

    Ross-Macdonald P, de Silva H, Guo Q, Xiao H, Hung C-Y, Penhallow B, et al. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther. 2008;7:3490–8.

    CAS  PubMed  Google Scholar 

  28. 28.

    May M, Schelle I, Brakebusch C, Rottner K, Genth H. Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry. Cell Cycle. 2014;13:2211–21.

    PubMed  Google Scholar 

  29. 29.

    Kumar R, Vadlamudi RK. Emerging functions of p21-activated kinases in human cancer cells. J Cell Physiol. 2002;193:133–44.

    CAS  PubMed  Google Scholar 

  30. 30.

    Davila M, Jhala D, Ghosh D, Grizzle WE, Chakrabarti R. Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer. Mol Cancer. 2007;6:40.

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sumi T, Hashigasako A, Matsumoto K, Nakamura T. Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition. Exp Cell Res. 2006;312:1021–30.

    CAS  PubMed  Google Scholar 

  32. 32.

    Moss DK, Betin VM, Malesinski SD, Lane JD. A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation. J Cell Sci. 2006;119:2362–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kueh AJ, Herold MJ. Using CRISPR/Cas9 technology for manipulating cell death regulators. Methods Mol Biol. 2016;1419:253–64.

    PubMed  Google Scholar 

  34. 34.

    Heck DE, Vetrano AM, Mariano TM, Laskin JD. UVB light stimulates production of reactive oxygen species: unexpected role for catalase. J Biol Chem. 2003;278:22432–6.

    CAS  PubMed  Google Scholar 

  35. 35.

    Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, et al. Lethal effect of the anti-Fas antibody in mice. Nature. 1993;364:806–9.

    CAS  PubMed  Google Scholar 

  36. 36.

    Mayer CT, Gazumyan A, Kara EE, Gitlin AD, Golijanin J, Viant C, et al. The microanatomic segregation of selection by apoptosis in the germinal center. Science. 2017;358:eaao2602.

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Levee MG, Dabrowska MI, Lelli JL, Hinshaw DB. Actin polymerization and depolymerization during apoptosis in HL-60 cells. Am J Physiol. 1996;271(6 Pt 1):C1981–92.

    CAS  PubMed  Google Scholar 

  38. 38.

    Sánchez-Alcázar JA, Rodríguez-Hernández Á, Cordero MD, Fernández-Ayala DJM, Brea-Calvo G, Garcia K, et al. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis. Apoptosis. 2007;12:1195–208.

    PubMed  Google Scholar 

  39. 39.

    Matsui T, Amano M, Yamamoto T, Chihara K, Nakafuku M, Ito M, et al. Rho‐associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 1996;15:2208–16.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Bianchi ME, Manfredi AA. High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity. Immunol Rev. 2007;220:35–46.

    CAS  PubMed  Google Scholar 

  41. 41.

    Chen G, Ward MF, Sama AE, Wang H. Extracellular HMGB1 as a proinflammatory cytokine. J Inter Cytokine Res. 2004;24:329–33.

    Google Scholar 

  42. 42.

    Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat Commun. 2017;8:14128.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Wang Y, Gao W, Shi X, Ding J, Liu W, He H, et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature. 2017;547:99–103.

    CAS  PubMed  Google Scholar 

  44. 44.

    Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 2009;16:3–11.

    CAS  PubMed  Google Scholar 

  46. 46.

    Arslan SY, Son K-N, Lipton HL. During infection, Theiler’s virions are cleaved by caspases and disassembled into pentamers. J Virol. 2016;90:3573–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Aprahamian T, Rifkin I, Bonegio R, Hugel B, Freyssinet J-M, Sato K, et al. Impaired clearance of apoptotic cells promotes synergy between atherogenesis and autoimmune disease. J Exp Med. 2004;199:1121–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Korb LC, Ahearn JM. C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited. J Immunol. 1997;158:4525–8.

    CAS  PubMed  Google Scholar 

  49. 49.

    Song J, Tan H, Shen H, Mahmood K, Boyd SE, Webb GI, et al. Cascleave: towards more accurate prediction of caspase substrate cleavage sites. Bioinformatics. 2010;26:752–60.

    CAS  PubMed  Google Scholar 

  50. 50.

    Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature. 1998;393:805–9.

    CAS  PubMed  Google Scholar 

  51. 51.

    Mills JC, Stone NL, Erhardt J, Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol. 1998;140:627–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Shiratsuchi A, Mori T, Nakanishi Y. Independence of Plasma Membrane Blebbing from Other Biochemical and Biological Characteristics of Apoptotic Cells 1. J Biochem. 2002;132:381–6.

    CAS  PubMed  Google Scholar 

  53. 53.

    Orlando KA, Pittman RN. Rho kinase regulates phagocytosis, surface expression of GlcNAc, and Golgi fragmentation of apoptotic PC12 cells. Exp Cell Res. 2006;312:3298–311.

    CAS  PubMed  Google Scholar 

  54. 54.

    Nakaya M, Tanaka M, Okabe Y, Hanayama R, Nagata S. Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages. J Biol Chem. 2006;281:8836–42.

    CAS  PubMed  Google Scholar 

  55. 55.

    Tosello-Trampont A-C, Nakada-Tsukui K, Ravichandran KS. Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling. J Biol Chem. 2003;278:49911–9.

    CAS  PubMed  Google Scholar 

  56. 56.

    Parnaik R, Raff MC, Scholes J. Differences between the clearance of apoptotic cells by professional and non-professional phagocytes. Curr Biol. 2000;10:857–60.

    CAS  PubMed  Google Scholar 

  57. 57.

    Phan TK, Lay FT, Hulett MD. Importance of phosphoinositide binding by human β-defensin 3 for Akt-dependent cytokine induction. Immunol Cell Biol. 2018;96:54–67.

    CAS  PubMed  Google Scholar 

  58. 58.

    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Diamond RA, Demaggio S. In Living Color: Protocols in Flow Cytometry and Cell Sorting. Berlin: Springer Berlin Heidelberg; 2000. p. 802.

  60. 60.

    Beletskii A, Cooper M, Sriraman P, Chiriac C, Zhao L, Abbot S, et al. High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye. Biotechniques. 2005;39:894–7.

    CAS  PubMed  Google Scholar 

  61. 61.

    Poon IK, Baxter AA, Lay FT, Mills GD, Adda CG, Payne JA, et al. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis. Elife. 2014;3:e01808.

    PubMed  PubMed Central  Google Scholar 

  62. 62.

    Atkin-Smith GK, Paone S, Zanker DJ, Duan M, Phan TK, Chen W, et al. Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting. Sci Rep. 2017;7:39846.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Rosenberg IM. Protein analysis and purification: benchtop techniques. Basel: Birkhäuser; 2005. p. 520.

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Acknowledgements

We would like to thank the LIMS BioImaging Facility for assistance with microscopy. We would also like to thank Alyce Mayfosh for assistance with quantitative PCR. This work was supported by grants from the National Health & Medical Research Council of Australia (GNT1125033 and GNT1140187) and Australian Research Council (DP170103790) to I.K.H.P.

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Correspondence to Ivan K. H. Poon.

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Tixeira, R., Phan, T.K., Caruso, S. et al. ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly. Cell Death Differ 27, 102–116 (2020). https://doi.org/10.1038/s41418-019-0342-5

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