Apoptosis is a genetically programmed energy-dependent process of cell demise, characterized by specific morphological and biochemical events in which the activation of caspases has an essential role. During apoptosis the cytoskeleton participates actively in characteristic morphological rearrangements of the dying cell. This reorganisation has been assigned mainly to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent reports have showed that microtubules are reformed during the execution phase of apoptosis organizing an apoptotic microtubule network (AMN). AMN is organized behind plasma membrane, forming a cortical structure. Apoptotic microtubules repolymerization takes place in many cell types and under different apoptotic inducers. It has been hypothesized that AMN is critical for maintaining plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disorganization leads apoptotic cells to secondary necrosis and the release of potential toxic molecules which can damage neighbor cells and promotes inflammation. Therefore, AMN formation during physiological apoptosis or in pathological apoptosis induced by anti-cancer treatments is essential for tissue homeostasis and the prevention of additional cell damage and inflammation. V C 2015Wiley Periodicals, Inc.Key Words: apoptosis; microtubules; actin; cytoskeleton Apoptosis A poptosis is an intracellular signaling pathway conserved across evolution dependent on a caspase-mediated proteolytic cascade that leads to a program of cell death through a series of cellular changes distinct of cell necrosis [Kerr et al., 1972]. Apoptosis plays a critical role in tissue remodeling during development, tissue homeostasis, cleaning of senescent cells, and removal of the cells with severe DNA damage [reviewed in Fuchs and Steller, 2011]. Given that cell necrosis causes the release of toxic molecules and causes inflammation [Savill et al., 2002], an important function of apoptosis is to isolate dying cells and prepare them for elimination by phagocytosis. This program of cell death is carried out by organelle-directed regulators, including the Bcl-2 proteins [Czabotar et al., 2014], and ultimately executed by cysteine proteases of the caspase family [Aslan and Thomas, 2009;McIlwain et al., 2015]. Some caspases, such caspase-8 and 29, operate as "initiators" and transmit cell-death signals by activating "executioner or effector" caspases, such as caspase-3, 26 and 27. After activation, "effector" caspases cleave a wide range of cellular proteins that are required for cell survival [Poreba et al., 2013]. Caspase activation can be initiated by extracellular stimuli as well as by alterations of intracellular homeostasis that often converge primarily or secondarily at mitochondria [Tait and Green, 2010]. Regardless of the precise molecular mechanisms leading to caspase activation, apoptotic cells exhibit characteristic features, eventually fragmenting into discre...

Apoptosis is a regulated energy-dependent process of cell death characterized by specific morphological and biochemical features in which caspase activation has a central role. During apoptosis, cells undergo characteristic morphological rearrangements in which the cytoskeleton participates actively. From a historical point of view, this reorganization has been assigned mainly to actinomyosin ring contraction with microtubule and intermediate filaments, both reported to be depolymerized at early stages of apoptosis. However, recent results have shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis, forming an apoptotic microtubule network (AMN). AMN is closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon." Apoptotic microtubules' reorganization has been reported in many cell types and under many apoptotic inducers. Recently, it has been proposed that AMN is essential for preserving plasma membrane permeability and cell morphology during the execution phase of apoptosis. Apoptotic microtubules' depolymerization leads cells to secondary necrosis and the release of toxic intracellular contents that can harm surrounding cells and initiate inflammation. Therefore, microtubules' reorganization in physiological apoptosis during development and in the adult organism or in pathological apoptosis induced by anticancer treatments or chronic inflammation is essential for tissue homeostasis, preventing cell damage and inflammation.

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. We were able by using a cocktail composed of taxol (a microtubule stabilizer), Zn (a caspase inhibitor) and coenzyme Q (a lipid antioxidant) to stabilize H460 apoptotic cells in cell cultures for at least 72hours preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cells characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels, plasma membrane potential, PS externalization and ability of being phagocytosed. Stabilized apoptotic cells can be considered as dying cells in which the cellular cortex and plasma membrane are maintained intact or alive. In a metaphorical sense, we can consider them as "living dead" or "zombie cells". Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.