Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

22 páginas, 15 figuras.
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.
This work was supported by FIS PI080500 grant, FIS EC08/00076 grant, Ministerio de Sanidad, Spain, and by AEPMI (Asociación de Enfermos de Patología Mitocondrial). This group is founded by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, and Fundación Española de Enfermedades Lisosomales (FEEL). Mario de la Mata is granted with a fellowship from Colegio Oficial de Farmacéuticos de Sevilla.