Skip to main content
SpringerLink
Log in

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional γ-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon”. Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Abbreviations

AMN:

apoptotic microtubule network

CPT:

camptothecin

COL:

colchicine

CYTO:

cytochalasin

EGTA-AM:

Ethyleneglycol-bis(b-aminoethyl)-N,N,N′,N′- tetraacetoxymethyl Ester

LDH:

lactic dehydrogenase

References

  1. Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257

    PubMed  CAS  Google Scholar 

  2. Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2(12):965–975

    Article  PubMed  CAS  Google Scholar 

  3. Wilson MR (1998) Apoptosis: unmasking the executioner. Cell Death Differ 5(8):646–652

    Article  PubMed  CAS  Google Scholar 

  4. Afford S, Randhawa S (2000) Apoptosis. Mol Pathol 53(2):55–63

    Article  PubMed  CAS  Google Scholar 

  5. Mills JC, Stone NL, Pittman RN (1999) Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J Cell Biol 146(4):703–708

    Article  PubMed  CAS  Google Scholar 

  6. Pittman S, Geyp M, Fraser M, Ellem K, Peaston A, Ireland C (1997) Multiple centrosomal microtubule organising centres and increased microtubule stability are early features of VP-16-induced apoptosis in CCRF-CEM cells. Leuk Res 21(6):491–499

    Article  PubMed  CAS  Google Scholar 

  7. Pittman SM, Strickland D, Ireland CM (1994) Polymerization of tubulin in apoptotic cells is not cell cycle dependent. Exp Cell Res 215(2):263–272

    Article  PubMed  CAS  Google Scholar 

  8. Moss DK, Lane JD (2006) Microtubules: forgotten players in the apoptotic execution phase. Trends Cell Biol 16(7):330–338

    Article  PubMed  CAS  Google Scholar 

  9. Moss DK, Betin VM, Malesinski SD, Lane JD (2006) A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation. J Cell Sci 119(Pt 11):2362–2374

    Article  PubMed  CAS  Google Scholar 

  10. Brea-Calvo G, Rodriguez-Hernandez A, Fernandez-Ayala DJ, Navas P, Sanchez-Alcazar JA (2006) Chemotherapy induces an increase in coenzyme Q10 levels in cancer cell lines. Free Radic Biol Med 40(8):1293–1302

    Article  PubMed  CAS  Google Scholar 

  11. Sanchez-Alcazar JA, Bradbury DA, Brea-Calvo G, Navas P, Knox AJ (2003) Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression. Apoptosis 8(6):639–647

    Article  PubMed  CAS  Google Scholar 

  12. Rodriguez-Hernandez A, Brea-Calvo G, Fernandez-Ayala DJ, Cordero M, Navas P, Sanchez-Alcazar JA (2006) Nuclear caspase-3 and caspase-7 activation, and Poly(ADP-ribose) polymerase cleavage are early events in camptothecin-induced apoptosis. Apoptosis 11(1):131–139

    Article  PubMed  CAS  Google Scholar 

  13. Sanchez-Alcazar JA, Khodjakov A, Schneider E (2001) Anticancer drugs induce increased mitochondrial cytochrome c expression that precedes cell death. Cancer Res 61(3):1038–1044

    PubMed  CAS  Google Scholar 

  14. Oakley BR, Akkari YN (1999) Gamma-tubulin at ten: progress and prospects. Cell Struct Funct 24(5):365–372

    Article  PubMed  CAS  Google Scholar 

  15. Andersen SSL (2000) Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18. Trends in Cell Biology 10(7):261–267

    Article  PubMed  CAS  Google Scholar 

  16. Tokuraku K, Katsuki M, Nakagawa H, Kotani S (1999) A new model for microtubule-associated protein (MAP)-induced microtubule assembly: the Pro-rich region of MAP4 promotes nucleation of microtubule assembly in vitro. Eur J Biochem 259(1):158–166

    Article  PubMed  CAS  Google Scholar 

  17. Tombal B, Denmeade SR, Gillis JM, Isaacs JT (2002) A supramicromolar elevation of intracellular free calcium ([Ca(2+)](i)) is consistently required to induce the execution phase of apoptosis. Cell Death Differ 9(5):561–573

    Article  PubMed  CAS  Google Scholar 

  18. Mattson MP, Chan SL (2003) Calcium orchestrates apoptosis. Nat Cell Biol 5(12):1041–1043

    Article  PubMed  CAS  Google Scholar 

  19. Bonanno E, Ruzittu M, Carla EC, Montinari MR, Pagliara P, Dini L (2000) Cell shape and organelle modification in apoptotic U937 cells. Eur J Histochem 44(3):237–246

    PubMed  CAS  Google Scholar 

  20. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3(4):339–345

    Article  PubMed  CAS  Google Scholar 

  21. Croft DR, Coleman ML, Li S, Robertson D, Sullivan T, Stewart CL et al (2005) Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. J Cell Biol 168(2):245–255

    Article  PubMed  CAS  Google Scholar 

  22. Rao L, Perez D, White E (1996) Lamin proteolysis facilitates nuclear events during apoptosis. J Cell Biol 135(6, Part 1):1441–1455

    Article  PubMed  CAS  Google Scholar 

  23. Caulin C, Salvesen GS, Oshima RG (1997) Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 138(6):1379–1394

    Article  PubMed  CAS  Google Scholar 

  24. Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL (2001) Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death And Differ 8(5):443–450

    Article  CAS  Google Scholar 

  25. Bonfoco E, Leist M, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P (1996) Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation. J Neurochem 67(6):2484–2493

    Article  PubMed  CAS  Google Scholar 

  26. Mills JC, Lee VM, Pittman RN (1998) Activation of a PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution phase of apoptosis. J Cell Sci 111(Pt 5):625–636

    PubMed  CAS  Google Scholar 

  27. Janmey PA (1998) The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol Rev 78(3):763–781

    PubMed  CAS  Google Scholar 

  28. Oshima RG (2002) Apoptosis and keratin intermediate filaments. Cell Death Differ 9(5):486–492

    Article  PubMed  CAS  Google Scholar 

  29. Job D, Valiron O, Oakley B (2003) Microtubule nucleation. Curr Opin Cell Biol 15(1):111–117

    Article  PubMed  CAS  Google Scholar 

  30. Adrain C, Duriez PJ, Brumatti G, Delivani P, Martin SJ (2006) The cytotoxic lymphocyte protease, granzyme B, targets the cytoskeleton and perturbs microtubule polymerization dynamics. J Biol Chem 281(12):8118–8125

    Article  PubMed  CAS  Google Scholar 

  31. Shiina N, Tsukita S (1999) Regulation of microtubule organization during interphase and M phase. Cell Struct Funct 24(5):385–391

    Article  PubMed  CAS  Google Scholar 

  32. Schwab BL, Guerini D, Didszun C, Bano D, Ferrando-May E, Fava E et al (2002) Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis. Cell Death Differ 9(8):818–831

    Article  PubMed  CAS  Google Scholar 

  33. Nicotera P, Orrenius S (1998) The role of calcium in apoptosis. Cell Calcium 23(2–3):173–180

    Article  PubMed  CAS  Google Scholar 

  34. Martin S, Reutelingsperger C, McGahon A, Rader J, van Schie R, LaFace D et al (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182(5):1545–1556

    Article  PubMed  CAS  Google Scholar 

  35. Fadok V, Voelker D, Campbell P, Cohen J, Bratton D, Henson P (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148(7):2207–2216

    PubMed  CAS  Google Scholar 

  36. Hampton MB, Vanags DM, Isabella Porn-Ares M, Orrenius S (1996) Involvement of extracellular calcium in phosphatidylserine exposure during apoptosis. FEBS Lett 399(3):277–282

    Article  PubMed  CAS  Google Scholar 

  37. Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM (1997) Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase. J Biol Chem 272(42):26159–26165

    Article  PubMed  CAS  Google Scholar 

  38. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM (2000) A receptor for phosphatidylserine-specific clearance of apoptotic cells. 405(6782):85–90

  39. Verhoven B, Schlegel R, Williamson P (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182(5):1597–1601

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Antonio Arroyo and John Pearson for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-CSIC, Carretera de Utrera Km 1, Sevilla, 41013, Spain

    José A. Sánchez-Alcázar, Ángeles Rodríguez-Hernández, Mario D. Cordero, Daniel J. M. Fernández-Ayala, Gloria Brea-Calvo, Katherina Garcia & Plácido Navas

Authors
  1. José A. Sánchez-Alcázar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ángeles Rodríguez-Hernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mario D. Cordero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel J. M. Fernández-Ayala
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gloria Brea-Calvo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katherina Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Plácido Navas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José A. Sánchez-Alcázar.

Electronic supplementary material

Figure S1

Fluorescence microscopy of microtubule in control, CPT, and staurosporine treated primary human fibroblasts. Cells were grown on glass coverslips and treated with CPT or staurosporin for 48 hours. Then cells were fixed and immunostained with mouse anti α-tubulin (red). Nuclear morphology was revealed by staining with Hoechst 33342. Arrows, tubulin reorganization in apoptotic cells. Bar: 30 μm

Figure S2

Fluorescence microscopy of microtubule in control, CPT, and TRAIL treated HeLa cells. Cells were grown on glass coverslips and treated with CPT or TRAIL for 48 hours. Cells were then fixed and immunostained with mouse anti α-tubulin (red). Nuclear morphology was revealed by staining with Hoechst 33342. Arrows, tubulin reorganization in apoptotic cells. Bar: 30 μm

Figure S3

Fluorescence microscopy of microtubule in, CPT, and serum withdrawal treated HeLa cells. Cells were grown on glass coverslips and treated with CPT or serum withdrawal for 48 hours. Cells were fixed and immunostained with mouse anti α-tubulin (red). Nuclear morphology was revealed by staining with Hoechst 33342. Arrows, tubulin reorganization in apoptotic cells. Bar: 30 mm

Figure S4

Fluorescence microscopy of microtubule in control H460 cells. Cells were grown on glass coverslips for 48 hours. Cells were then fixed and immunostained with mouse anti β-tubulin (green). Nuclear morphology was revealed by staining with Hoechst 33342. Arrows, tubulin reorganization in apoptotic cells. Bar = 15 μm.

Figure S5

Light and fluorescence microscopy of CPT-treated H460 apoptotic cells showing AMN, surrounding the cell beneath the plasma membrane. Cells were grown on glass coverslips for 48 hours. Cells were then fixed and immunostained with mouse anti α-tubulin. Nuclear morphology was revealed by staining with Hoechst 33342 Arrow: apoptotic cell.

Figure S6

Light and fluorescence microscopy CPT-treated H460 apoptotic cell showing the AMN extending from body of the cell into slender spikes. Cells were grown on glass coverslips for 48 hours. Cells were then fixed and immunostained with mouse anti β-tubulin (red). Nuclear morphology was revealed by staining with Hoechst 33342.

Figure S7

Fluorescence microscopy of CPT-treated LLCPK-1α cells. Cells were grown on glass coverslips and treated with CPT for 12 hours. Cells were then fixed. Nuclear morphology was revealed by staining with Hoechst 33342. White arrow: apoptotic body with the AMN enclosing a small nuclear fragment. Yellow arrow: large cellular fragment with an AMN.

Figure S8

Fluorescence microscopy of CPT+zVAD treated H460 apoptotic cell showing the AMN in the presence of cytochrome c release (arrow). Cells were grown on glass coverslips for 48 hours. Cells were then fixed and immunostained with rabbit anti α-tubulin (red) and mouse anti cytochrome c. Nuclear morphology was revealed by staining with Hoechst 33342 Nuclei are condensed but not fragmented due to caspase inactivation. Panel A, Bar: 30 mm. Panel B, Bar: 15 μm.

Figure S9

Light and fluorescence microscopy of CPT treated H460 apoptotic cell showing AMN formation and some spots of polymerized actin at the end of the spikes. Cells were grown on glass coverslips for 48 hours. Cells were then fixed and immunostained with mouse anti α-tubulin (green) and TRITC-phalloidin to visualize actin filaments (red). Nuclear morphology was revealed by staining with Hoechst 33342.

Figure S10

Fluorescence microscopy of control and CPT-treated LLCPK-1α cells. Cells were grown on glass coverslips and treated with CPT for 12 hours. Cells were then fixed and stained with and TRITC-phalloidin to visualize actin filaments (red) (red). Nuclear morphology was revealed by staining with Hoechst 33342. Arrow: actin spots in an apoptotic cell with AMN.

Figure S11

LDH release in the medium of Control, CPT, CPT + CYTO, CPT + COL Control + CYTO (X), and Control + COL treated HeLa cells. Cells were treated as described in Material and methods. *, p<0,01, between CPT + COL and CPT treated cells (n=3).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sánchez-Alcázar, J.A., Rodríguez-Hernández, Á., Cordero, M.D. et al. The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis. Apoptosis 12, 1195–1208 (2007). https://doi.org/10.1007/s10495-006-0044-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-006-0044-6

Keywords

Search

Navigation