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Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis

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Abstract

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.

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Abbreviations

AMN:

Apoptotic microtubule network

CPT:

Camptothecin

CYTO:

Cytochalasin

ΔΨm:

Mitochondria membrane potential

FAK:

Focal adhesion kinase

GADPH:

Glyceraldehyde-3-phosphate dehydrogenase

LDH:

Lactic dehydrogenase

PMCA-4:

Plasma membrane Ca2+ ATPase

PS:

Phosphadidylserine

ROCK-1:

Rho-associated, coiled-coil containing protein kinase 1

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Acknowledgments

This work was supported by FIS PI10/00543 Grant, FIS EC08/00076 Grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS 111242 Grant, Servicio Andaluz de Salud-Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, and by Asociación de Enfermos de Patología Mitocondrial (AEPMI).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas (CSIC), Universidad Pablo de Olavide, and Centro de Investigación Biomédica en Red: Enfermedades Raras, Instituto de Salud Carlos III, Carretera de Utrera Km 1, 41013, Seville, Spain

    Manuel Oropesa-Ávila, Alejandro Fernández-Vega, Mario de la Mata, Juan Garrido-Maraver, David Cotán, Marina Villanueva Paz, Ana Delgado Pavón, Elizabet Alcocer-Gómez, Isabel de Lavera, Rafael Lema & José A. Sánchez-Alcázar

  2. Facultad de Odontología, Universidad de Sevilla, 41009, Seville, Spain

    Mario D. Cordero

  3. Sistemas Físicos, Químicos y Naturales-Universidad Pablo de Olavide, 41013, Seville, Spain

    Ana Paula Zaderenko

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  1. Manuel Oropesa-Ávila
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  2. Alejandro Fernández-Vega
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  12. Ana Paula Zaderenko
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  13. José A. Sánchez-Alcázar
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Correspondence to José A. Sánchez-Alcázar.

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Oropesa-Ávila, M., Fernández-Vega, A., de la Mata, M. et al. Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis. Apoptosis 19, 1364–1377 (2014). https://doi.org/10.1007/s10495-014-1015-y

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