Article | Published:

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

  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.

  3. 3.

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

  4. 4.

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

  5. 5.

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

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  14. 14.

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

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  26. 26.

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

  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.

  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.

  29. 29.

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

  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.

  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.

  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.

  33. 33.

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

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  41. 41.

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

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  63. 63.

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

Download references

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.

Author information

Correspondence to Ivan K. H. Poon.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by S. Kumar

Supplementary information

Supplementary Figures

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5