title: Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.
abstract: Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.
authors.affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Carretera de Utrera Km. 1, Seville, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Oropesa
authors.forename: Mario
authors.initials: M
authors.lastname: de la Mata
authors.forename: Juan Garrido
authors.initials: JG
authors.lastname: Maraver
authors.forename: Mario D
authors.initials: MD
authors.lastname: Cordero
authors.forename: David
authors.initials: D
authors.lastname: Cotán
authors.forename: Angeles
authors.initials: A
authors.lastname: Rodríguez-Hernández
authors.forename: Irene
authors.initials: I
authors.lastname: Domínguez-Moñino
authors.forename: Manuel
authors.initials: M
authors.lastname: de Miguel
authors.forename: Plácido
authors.initials: P
authors.lastname: Navas
authors.forename: José A
authors.initials: JA
authors.lastname: Sánchez-Alcázar
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D045744:Cell Line, Tumor; D003078:Colchicine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D000199:Actins; D009840:Oligomycins; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D006180:Proton-Translocating ATPases; D003078:Colchicine; D002118:Calcium