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Apoptosis

, Volume 24, Issue 11–12, pp 862–877 | Cite as

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis

  • Sarah Caruso
  • Georgia K. Atkin-Smith
  • Amy A. Baxter
  • Rochelle Tixeira
  • Lanzhou Jiang
  • Dilara C. Ozkocak
  • Jascinta P. Santavanond
  • Mark D. Hulett
  • Peter Lock
  • Thanh Kha Phan
  • Ivan K. H. PoonEmail author
Article

Abstract

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Keywords

Apoptotic bodies Apoptotic cell disassembly Apoptotic morphology Apoptopodia Cytoskeletal components Membrane protrusions 

Notes

Acknowledgements

We thank the La Trobe BioImaging Platform for access to microscopy and flow cytometry equipment and assistance with microscopy. We thank Dr Hendrika Duivenvoorden for her assistance with 3D cultures. This work was supported by grants from the National Health & Medical Research Council of Australia (GNT1141732, GNT1125033, GNT1140187), Australian Research Council (DP170103790) and La Trobe University (RFA2018).

Author contributions

SC, GKAS and IKHP designed and performed experiments with assistance from co-authors. AB generated and performed experiments on vimentin deficient cells. SC, GKAS and IKHP wrote the manuscript with input from co-authors.

Compliance with ethical standards

Competing interests

The authors declare no competing financial interests.

Supplementary material

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References

  1. 1.
    Mattila PK, Lappalainen P (2008) Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9:446–454PubMedGoogle Scholar
  2. 2.
    Small JV et al (2002) The lamellipodium: where motility begins. Trends Cell Biol 12:112–120PubMedGoogle Scholar
  3. 3.
    Davis DM, Sowinski S (2008) Membrane nanotubes: dynamic long-distance connections between animal cells. Nat Rev Mol Cell Biol 9:431–436PubMedGoogle Scholar
  4. 4.
    Eddy RJ et al (2017) Tumor cell invadopodia: invasive protrusions that orchestrate metastasis. Trends Cell Biol 27:595–607PubMedPubMedCentralGoogle Scholar
  5. 5.
    Ballestrem C et al (2000) Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration. Mol Biol Cell 11:2999–3012PubMedPubMedCentralGoogle Scholar
  6. 6.
    Schoumacher M et al (2010) Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia. J Cell Biol 189:541–556PubMedPubMedCentralGoogle Scholar
  7. 7.
    Rustom A et al (2004) Nanotubular highways for intercellular organelle transport. Science 303:1007–1010PubMedGoogle Scholar
  8. 8.
    Pollard TD, Cooper JA (2009) Actin, a central player in cell shape and movement. Science 326:1208–1212PubMedPubMedCentralGoogle Scholar
  9. 9.
    Keller KE et al (2017) Tunneling nanotubes are novel cellular structures that communicate signals between trabecular meshwork cells. Invest Ophthalmol Vis Sci 58:5298–5307PubMedPubMedCentralGoogle Scholar
  10. 10.
    Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465Google Scholar
  11. 11.
    Insall RH, Machesky LM (2009) Actin dynamics at the leading edge: from simple machinery to complex networks. Dev Cell 17:310–322PubMedGoogle Scholar
  12. 12.
    Sheetz MP, Wayne DB, Pearlman AL (1992) Extension of filopodia by motor-dependent actin assembly. Cell Motil Cytoskelet 22:160–169Google Scholar
  13. 13.
    Hanna SJ et al (2017) The role of Rho-GTPases and actin polymerization during macrophage tunneling nanotube biogenesis. Sci Rep 7:8547PubMedPubMedCentralGoogle Scholar
  14. 14.
    Isogai T et al (2015) Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex. J Cell Sci 128:3796–3810PubMedGoogle Scholar
  15. 15.
    Vinzenz M et al (2012) Actin branching in the initiation and maintenance of lamellipodia. J Cell Sci 125:2775–2785PubMedGoogle Scholar
  16. 16.
    Svitkina TM, Borisy GG (1999) Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol 145:1009–1026PubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang YL (1985) Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol 101:597–602PubMedGoogle Scholar
  18. 18.
    Bugyi B, Carlier MF (2010) Control of actin filament treadmilling in cell motility. Annu Rev Biophys 39:449–470PubMedGoogle Scholar
  19. 19.
    Euteneuer U, Schliwa M (1984) Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules. Nature 310:58–61PubMedGoogle Scholar
  20. 20.
    Mitchison T, Kirschner M (1988) Cytoskeletal dynamics and nerve growth. Neuron 1:761–772PubMedGoogle Scholar
  21. 21.
    Smith SJ (1988) Neuronal cytomechanics: the actin-based motility of growth cones. Science 242:708–715PubMedGoogle Scholar
  22. 22.
    Martins GG, Kolega J (2012) A role for microtubules in endothelial cell protrusion in three-dimensional matrices. Biol Cell 104:271–286PubMedGoogle Scholar
  23. 23.
    Atkin-Smith GK, Poon IK (2016) Disassembly of the dying: mechanisms and functions. Trends Cell Biol 27:151–162PubMedGoogle Scholar
  24. 24.
    Atkin-Smith GK et al (2015) A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nat Commun 6:7439PubMedPubMedCentralGoogle Scholar
  25. 25.
    Moss DK et al (2006) A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation. J Cell Sci 119:2362–2374PubMedPubMedCentralGoogle Scholar
  26. 26.
    Poon IKH et al (2014) Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature 507:329–334PubMedPubMedCentralGoogle Scholar
  27. 27.
    Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257PubMedPubMedCentralGoogle Scholar
  28. 28.
    Tixeira R et al (2017) Defining the morphologic features and products of cell disassembly during apoptosis. Apoptosis 22:475–477PubMedGoogle Scholar
  29. 29.
    Mills JC et al (1998) Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 140:627–636PubMedPubMedCentralGoogle Scholar
  30. 30.
    Sebbagh M et al (2001) Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3:346–352PubMedGoogle Scholar
  31. 31.
    Huot J et al (1998) SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. J Cell Biol 143:1361–1373PubMedPubMedCentralGoogle Scholar
  32. 32.
    Coleman ML et al (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3:339–345PubMedGoogle Scholar
  33. 33.
    Tixeira R et al (2019) ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly. Cell Death Differ.  https://doi.org/10.1038/s41418-019-0342-5 CrossRefPubMedGoogle Scholar
  34. 34.
    Kolomeisky AB, Fisher ME (2001) Force-velocity relation for growing microtubules. Biophys J 80:149–154PubMedPubMedCentralGoogle Scholar
  35. 35.
    Brangbour C et al (2011) Force-velocity measurements of a few growing actin filaments. PLoS Biol 9:e1000613PubMedPubMedCentralGoogle Scholar
  36. 36.
    Battaglia RA et al (2018) Vimentin on the move: new developments in cell migration. F1000Res 7:1796Google Scholar
  37. 37.
    Lanier MH, Kim T, Cooper JA (2015) CARMIL2 is a novel molecular connection between vimentin and actin essential for cell migration and invadopodia formation. Mol Biol Cell 26:4577–4588PubMedPubMedCentralGoogle Scholar
  38. 38.
    Chekeni FB et al (2010) Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis. Nature 467:863–867PubMedPubMedCentralGoogle Scholar
  39. 39.
    Poon IKH et al (2019) Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell-derived extracellular vesicles in vitro. J Extracell Vesicles 8:1608786PubMedPubMedCentralGoogle Scholar
  40. 40.
    Loitto VM et al (2007) Filopodia are induced by aquaporin-9 expression. Exp Cell Res 313:1295–1306PubMedGoogle Scholar
  41. 41.
    Karlsson T et al (2013) Fluxes of water through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane protrusions. PLoS One 8:e59901PubMedPubMedCentralGoogle Scholar
  42. 42.
    Breitsprecher D et al (2011) Cofilin cooperates with fascin to disassemble filopodial actin filaments. J Cell Sci 124:3305–3318PubMedPubMedCentralGoogle Scholar
  43. 43.
    Lane JD, Allan VJ, Woodman PG (2005) Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells. J Cell Sci 118:4059–4071PubMedGoogle Scholar
  44. 44.
    Sanchez-Alcazar JA et al (2007) The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis. Apoptosis. 12: 1195–1208PubMedGoogle Scholar
  45. 45.
    Jiang L et al (2017) Determining the contents and cell origins of apoptotic bodies by flow cytometry. Sci Rep 7:14444PubMedPubMedCentralGoogle Scholar
  46. 46.
    Onfelt B et al (2004) Cutting edge: Membrane nanotubes connect immune cells. J Immunol 173:1511–1513PubMedGoogle Scholar
  47. 47.
    Heckman CA, Plummer HK 3rd (2013) Filopodia as sensors. Cell Signal 25:2298–2311PubMedGoogle Scholar
  48. 48.
    Berda-Haddad Y et al (2011) Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha. Proc Natl Acad Sci USA 108:20684–20689PubMedGoogle Scholar
  49. 49.
    Brock CK et al (2019) Stem cell proliferation is induced by apoptotic bodies from dying cells during epithelial tissue maintenance. Nat Commun 10:1044PubMedPubMedCentralGoogle Scholar
  50. 50.
    Ma Q et al (2019) Mature osteoclast-derived apoptotic bodies promote osteogenic differentiation via RANKL-mediated reverse signaling. J Biol Chem 294:11240–11247PubMedGoogle Scholar
  51. 51.
    Zhu Z et al (2017) Macrophage-derived apoptotic bodies promote the proliferation of the recipient cells via shuttling microRNA-221/222. J Leukoc Biol 101:1349–1359PubMedPubMedCentralGoogle Scholar
  52. 52.
    Jiang L et al (2016) Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat Protoc 11:655–663PubMedGoogle Scholar
  53. 53.
    Kueh AJ, Herold MJ (2016) Using CRISPR/Cas9 technology for manipulating cell death regulators. Methods Mol Biol 1419:253–264PubMedGoogle Scholar
  54. 54.
    Hsu PD et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019
corrected publication 2019

Authors and Affiliations

  • Sarah Caruso
    • 1
  • Georgia K. Atkin-Smith
    • 1
  • Amy A. Baxter
    • 1
  • Rochelle Tixeira
    • 1
  • Lanzhou Jiang
    • 1
  • Dilara C. Ozkocak
    • 1
  • Jascinta P. Santavanond
    • 1
  • Mark D. Hulett
    • 1
  • Peter Lock
    • 1
  • Thanh Kha Phan
    • 1
  • Ivan K. H. Poon
    • 1
    Email author
  1. 1.Department of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceLa Trobe UniversityMelbourneAustralia

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