Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis M Oropesa-A ´vila 1 , A Ferna ´ndez-Vega 1 , M de la Mata 1 , JG Maraver 1 , MD Cordero 2 , D Cota ´n 1 , M de Miguel 2 , CP Calero 1 , MV Paz 1 , AD Pavo ´n 1 ,MASa ´nchez 1 , AP Zaderenko 3 , P Ybot-Gonza ´lez 4 and JA Sa ´nchez-Alca ´zar* ,1 Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as a-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit b4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na þ /Ca 2þ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na þ /K þ pump subunit bwas associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis. Cell Death and Disease (2013) 4, e527; doi:10.1038/cddis.2013.58; published online 7 March 2013 Subject Category: Experimental Medicine Apoptosis is a physiologic mechanism employed by most multicellular organisms to maintain tissue remodeling during development, tissue homeostasis, removal of senescent cells and the elimination of cells with severe genetic damage. 1 Moreover, many antineoplasic drugs such as campothecin, a topoisomerase I inhibitor, kill tumor cells by inducing apoptosis. This mechanism of cell death is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells. In apoptosis, all the degradative processes are isolated from the extracellular space by the plasma membrane, which remains impermeable. However, the mechanisms of how plasma membrane and associated proteins are protected from the action of caspases are not known. In contrast, necrosis is accompanied by disruption of plasma membrane integrity so all intracellular compounds are released to the intercellular space, thus inducing inflammation and more toxic effects to adjacent cells. 2,3 In contrast to apoptotic cells in vivo, cells which undergo apoptosis in vitro cannot usually be cleared by phagocytes and undergo a late process of secondary necrosis defined as a loss of cell membrane integrity, calcium influx from the medium and release of cell content into the extracellular space. 4 Previous evidence suggests that the actomyosin cytoskeleton has an essential role in apoptotic cell remodeling during the early 1 Centro Andaluz de Biologı ´a del Desarrollo (CABD-CSIC-Universidad Pablo de Olavide), and Centro de Investigacio ´n Biome ´dica en Red: Enfermedades Raras, Instituto de Salud Carlos III, Sevilla, Spain; 2 Departamento de Citologı ´a e Histologı ´a Normal y Patolo ´gica, Facultad de Medicina. Universidad de Sevilla, Sevilla, Spain; 3 Sistemas Fı ´sicos, Quı ´micos y Naturales-Universidad Pablo de Olavide, Sevilla, Spain and 4 Instituto de Biomedicina de Sevilla (IBIS)-CSIC, Hospital Virgen del Rocı ´o, Sevilla, Spain *Corresponding author: JA Sa ´nchez Alca ´zar, Centro Andaluz de Biologı ´a del Desarrollo (CABD). Consejo Superior de Investigaciones Cientı ´ficas. Universidad Pablo de Olavide, Carretera de Utrera Km 1, Sevilla 41013, Spain. Tel: 349 5497 8071; Fax: 349 5434 9376; E-mail: [email protected] Received 7.1.13; revised 4.2.13; accepted 5.2.13; Edited by A Stephanou Keywords: apoptosis; microtubules; cytoskeleton; caspases; secondary necrosis Abbreviations: AMN, apoptotic microtubule network; CPT, camptothecin; COL, colchicine; CYTO, cytochalasin; Dcm, mitochondria membrane potential; FAK, focal adhesion kinase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactic dehydrogenase; NCX, Na þ -Ca þ exchanger; PMCA-4, plasma membrane Ca 2þ ATPase; PS, Phosphatidylserine; ROCK-1, Rho-associated, coiled-coil containing protein kinase 1 Citation: Cell Death and Disease (2013) 4, e527; doi:10.1038/cddis.2013.58 & 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13 www.nature.com/cddis events of the execution phase, whereas all other cytoskeleton elements (microtubules and intermediate filaments) are dismantled. 5 However, during the course of the execution phase, the actomyosin filaments are also depolymerized by a caspase-dependent mechanism. In this situation apoptotic cell formed a network of apoptotic microtubules as the main cytoskeleton element of the apoptotic cell. The presence of microtubules in apoptotic cells has previously been reported. 6,7 Moreover, more recent results indicate that microtubules during apoptosis assist in the dispersal of nuclear and cellular fragments, 8,9 and may help to preserve the integrity of plasma membrane of the dying cell. 10 The aim of this study was to examine the role of AMN in preserving plasma membrane integrity during the execution phase of apoptosis. Our results suggest that AMN works as a physical barrier keeping an active caspase free area in the cellular cortex of apoptotic cells, and thus avoiding the cleavage of essential proteins in maintaining plasma membrane integrity. Results AMN works as a physical barrier against active caspases. To examine the disposition of microtubules during apoptosis and its relationship with plasma membrane, cells were fixed and stained for b-tubulin, plasma membrane and active caspase-3 (Figures 1a and b). Both attached apoptotic cells and floating cells, which represent apoptotic cells in secondary necrosis were analyzed. We also examined apoptotic cells treated with colchicine for 1 h or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited (Figures 1a and b). In apoptotic cells, AMN seemed to work as a physical barrier impeding active caspases to reach the cellular cortex and plasma membrane. The cellular cortex, which appears as a narrow area between the plasma membrane and the apoptotic microtubules was free of active caspase-3. On the contrary when AMN was depolymerized by colchicine, active caspase-3 reached freely the cellular cortex and plasma membrane. Likewise, the active caspase-3 free area in the cellular cortex disappeared in apoptotic cells treated with colchicine and the pan-caspase inhibitor z-VAD and in floating death cells without AMN that represent cells in secondary necrosis. As expected, control cells without treatment and control cells treated with colchicine for 1 h were not apoptotic and did not show activation of caspase-3. For a better visualization of AMN structure in genuine apoptotic cells we performed a 3D reconstruction with the Delta Visio ´n software of cells stained for microtubules, actin and nuclei. We found that apoptotic cells with fragmented nuclei lacked actin microfilaments and AMN presented the typical ‘cocoon’ like structure that may function as a protective barrier surrounding the whole-cell (Supplementary Figure S1). AMN prevents plasma membrane permeability. To assess the significance of the AMN in genuine apoptotic cells versus apoptotic cells without AMN, we examined plasma membrane permeability in both apoptotic cells and cells in secondary necrosis. We also analyzed apoptotic cells treated with colchicine for 1 h or colchicine plus the pancaspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Cells were examined using the Dead Red reagent, a red fluorescent nucleic acid stain that only labels permeable cells, thus testing plasma membrane integrity. 11 We found that AMN was present in almost 100% of adherent apoptotic cells that were impermeable to the supravital dye (Figures 2a and b). However, we observed that membrane integrity was impaired in secondary necrotic cells, in which AMN was disorganized (Figures 2a and b). Interestingly, membrane permeability was also impaired in apoptotic cells, in which AMN was disorganized after colchicine treatment. However, plasma membrane remained impermeable in apoptotic cells treated simultaneously with colchicine and z-VAD, suggesting that although active caspases were able to reach the cellular cortex after AMN disorganization by colchicine, functional active caspases and consequently cleavage of cellular cortex and plasma membrane proteins are necessary to impair plasma membrane permeability. As expected, control cells without treatment and control cells treated with colchicine for 1 h remained impermeable. These observations were quantified scoring the proportion of living (impermeable and non-apoptotic nuclei), apoptotic (impermeable and fragmented nuclei) and secondary necrotic cells (permeable and fragmented nuclei) under the different experimental conditions (Figure 2b). Plasma membrane and cell cortex proteins are protected from caspase-mediated cleavage by AMN. To verify the functional relevance of AMN as a physical barrier during the execution phase of apoptosis, we examined the integrity of well-known cytosolic, nuclear, cellular cortex and plasma membrane proteins which are common substrates of effector caspases, in control, apoptotic and secondary necrotic cells. Apoptotic cells were also treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD for 1 h to examine the effect of AMN depolymerization when caspases were active or inhibited (Figure 3). Western blot analysis revealed that initiator caspase-9 and efector caspases-3 and -7 were activated and nuclear (PARP and lamin B) and cytosolic proteins (cytokeratins 5/8, cytokeratin 18 and ROCK-1) were cleaved in genuine apoptotic cells with AMN indicating that caspases were degrading nuclear and cytosolic components during the execution phase of apoptosis. On the contrary, cellular cortex and plasma membrane proteins such as a-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin, integrin b4, plasma membrane Ca 2þ ATPase (PMCA-4), Na þ -Ca þ exchanger (NCX) and Na þ /K þ pump subunit b, were intact in apoptotic cells with AMN. However, cellular cortex and transmembrane proteins were cleaved when AMN was disorganized in apoptotic cells by colchicine treatment or in secondary necrotic cells. As an exception, the plasma membrane protein Na þ /K þ pump subunit a,was not cleaved after AMN disorganization, suggesting that this particular subunit has no cleavage sequence for caspases. The critical role of caspase-mediated cleavage of cellular cortex and transmembrane proteins after AMN depolimerization was demonstrated because their degradation was AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 2 Cell Death and Disease prevented when z-VAD was added simultaneously with colchicine. All together these data indicate that AMN in apoptotic cells encloses most of the cellular volume where active caspases are degrading cytosolic and nuclear proteins, keeping the cellular cortex and plasma membrane free of active caspases. To confirm these data, we performed immunofluorescence analysis of cellular cortex and plasma membrane proteins to visualize its integrity in the presence or absence of AMN. As is shown in Figure 4, PMCA-4 is a plasma membrane protein involved in calcium extrusion preserved in apoptotic cells with AMM but it was degraded when AMN was depolymerized by 1 h incubation with colchicine or in cells in secondary necrosis without AMN. PMCA-4 cleavage after AMN depolimerization was mediated by caspases because its degradation was prevented when z-VAD was added simultaneously with colchicine (Figure 4). Likewise, FAK, another cellular cortex protein involved in cell adhesion complexes, was preserved in apoptotic cells Active Caspase-3 aTubulin βHoechst WG-agglutinin Merge Apoptotic Apoptotic +COL Secondary necrosis Control Control +COL Apoptotic +COL +Z-vad AMN ACTIVE CASPASE-3 AMN depolymerization Apoptotic bApoptotic + COL ACTIVE CASPASE-3 Plasma Membrane Plasma Membrane Figure 1 AMN during the execution phase of apoptosis. (a) Fluorescence microscopy of microtubules, plasma membrane and active caspase-3 in control cells, control cells treated with 2 mMcolchicine (Control þCOL), apoptotic cells, apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mMz-VAD (Apoptotic þCOL þz-VAD) for 1 h and cells in secondary necrosis. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment and cytospined on a slide. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Then, cells were fixed and immunostained with anti-b-tubulin (green), anti-active caspase-3. Plasma membrane was revealed by staining with WG-agglutinin. Nuclear morphology was revealed by staining with Hoechst 33342 (1 mg/ml). Bar ¼15 mm. (b) Magnification of apoptotic cells and apoptotic cells after 1 h of treatment with colchicine AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 3 Cell Death and Disease Apoptotic +COL Control Apoptotic +COL +Z-VAD Apoptotic SECONDARY NECROSIS Hoechst Permeability Merge Control +COL Percentage (%) 0 10 20 30 40 50 60 70 80 90 100 Control living apoptotic necrosis Apoptotic Apoptotic + COL Apoptotic +COL +Z-VAD Secondary Necrosis * *a *a b * a c *a *a b * a c Control + COL Tubulin  a b Figure 2 AMN and plasma membrane permeability. (a) Fluorescence microscopy of microtubules and plasma membrane permeability in control cells, control cells treated with 2 mMcolchicine (Control þCOL), apoptotic cells, apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mMz-VAD (Apoptotic þ COL þz-VAD) for 1 h and cells in secondary necrosis. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment and cytospined on a slide. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Before fixation cells were treated with the Dead Red dye, then fixed and immunostained with mouse anti-b-tubulin (green). Nuclear morphology was revealed by staining with Hoechst 33342 (1 mg/ml). Bar ¼15 mm. (b) Proportion of living (black), apoptotic (hatched) and secondary necrosis (white) H460 cells, based on the cell and nuclear morphological characteristics and plasma membrane permeability. More than 100 cells were examined for each experimental condition. *Po0.01 significant differences with respect to Control cells. a Po0.01, significant differences with respect to Apoptotic cells. b Po0.01, significant differences with respect to Apoptotic þCOL. c Po0.01, significant differences with respect to Apoptotic þCOL þz-VAD treated cells AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 4 Cell Death and Disease with AMM but it was degraded when AMN was depolymerized by colchicine or in cells in secondary necrosis without AMN (Supplementary Figure S2). Functional relevance of AMN in cell area and cell attachment. To examine whether AMN disorganization by colchicine, which allows the caspase-dependent cleavage of plasma membrane-cytoskeleton proteins such as spectrin and cell adhesion proteins such as paxilin, integrin b4, FAK and E-cadherin, was associated with cell collapse and substrate detachment, we measured cell area at the cell maximum perimeter and scored attached and floating cells in the medium in apoptotic cells with and without AMN. We found that AMN disorganization by 1 h of colchicine treatment induced a dramatic decreased in cell area and a significant increase in the number of floating cells (Figures 5a and b). Substrate detachment was prevented significantly in apoptotic cells after AMN disorganization by colchicine when caspases were inhibited by z-VAD suggesting the essential role of caspase-mediated cleavage of plasma membrane and cellular cortex proteins in this process. However, z-VAD was not able to prevent cell collapse after AMN depolymerization indicating that apoptotic microtubules per se are essential to maintain cell shape and morphology in apoptotic cells (Figure 5b). Likewise, colchicine treatment in control cells had not effect on cell attachment but it induced a significant reduction of cell area indicating that microtubules have an essential role in normal cell shape and morphology. Cell content leakage, calcium and sodium overload, and bioenergetics collapse are prevented by AMN. To confirm whether AMN was responsible for maintaining cellular integrity in apoptotic cells, we measured LDH release (a cytosolic enzyme used as intracellular marker), intracellular calcium and sodium levels, mitochondrial membrane potential (Dcm) and ATP levels in control, apoptotic and secondary necrotic cells. Apoptotic cells were also treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD for 1 h to examine the effect of AMN depolymerization when caspases were active or inhibited. Figure 6a shows that LDH release was significantly increased in the culture medium of apoptotic cells after AMN disorganization by colchicine treatment and in secondary necrotic cells, indicating that the absence of AMN allows the release of cell content. Interestingly, LDH release was prevented significantly in apoptotic cells without AMN by colchicine treatment when caspases were inhibited by z-VAD suggesting that caspase-mediated cleavage of plasma membrane and cellular cortex proteins was essential for releasing cell content. Previously, it has been demonstrated that caspases cleave and inactivate two key proteins in calcium homeostasis, PMCA-4 and NCX, in cells undergoing apoptosis. 12,13 PMCA-4 and NCX cleavage impairs intracellular Ca 2þ handling, which results in Ca 2þ overload and secondary necrosis. To confirm this hypothesis and to demonstrate the protective role of AMN during apoptosis, we measured intracellular calcium levels by FLUO-4 staining. Genuine apoptotic cells with AMN showed a moderate increase of FLUO-4 fluorescence respect to control cells. However, apoptotic cells without AMN by colchicine treatment or cells in secondary necrosis showed a notably increase of FLUO-4 fluorescence suggesting calcium overload (Figure 6b). Interestingly, calcium overload was significantly abolished in apoptotic cells without AMN by colchicine treatment but in PMCA Spectrin Cytokeratin 18 Active Caspase-3 Cleaved PMCA Cleaved spectrin Cytokeratin 5/8 E-cadherin Cleaved PARP GADPH NUCLEAR PROTEINS CYTOSOL PROTEINS FAK Cleaved FAK Active Caspase-7 Caspase-9 A ctive caspase-9 Lamin B ACTIVE CASPASES +z-VAD +COL Apoptotic +COL Control +COL Apoptotic Secondary NecrosisControl Apoptotic ROCK-1 CELULAR CORTEX PROTEINS: ACTIVE CASPASE FREE AREA WITH AMN NCX Integrin β4 Paxilin Na+/K+Pump α subunit Na+/K+ Pump β subunit Cleaved Paxilin Figure 3 Western blotting analysis of nuclear, cytosolic and cellular cortex proteins in the execution phase of apoptosis. Western blotting of nuclear (PARP, lamin C), cytosolic (cytokeratin 5/8, cytokeratin 18 and ROCK-1) and cellular cortex proteins (spectrin, FAK, paxilin, integrin b4, E-cadherin, PMCA-4, NCX, anti-Na þ / K þ pump subunit aand b, in control cells, control cells treated with 2 mMcolchicine (Control þCOL), apoptotic cells, apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mMz-VAD (Apoptotic þCOL þz- VAD) for 1 h and secondary necrotic cells. Apoptosis in H460 cells was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Cell lysates from H460 cells were resolved by SDS-PAGE and protein levels and cleavage fragments were determined by western blotting. Equal loading and transfer were shown by repeat probing with anti-GADPH AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 5 Cell Death and Disease which caspases were inhibited by z-VAD, suggesting that the activity of caspases were critical for PMCA-4 and NCX inactivation and subsequent calcium overload typical features of cells undergoing secondary necrosis. Likewise, cleavage of Na þ /K þ pump subunit bmay alter sodium extrusion and increase intracellular sodium levels. Apoptotic cells with AMN showed a moderate increase of sodium levels respect to control cells. However, apoptotic cells without AMN by colchicine treatment or cells in secondary necrosis showed a notably increase of sodium levels (Figure 6c). Sodium levels were significantly reduced in apoptotic cells devoid of AMN by colchicine treatment but in which caspases were inhibited by z-VAD, suggesting that the activity of caspases were critical for Na þ /K þ pump subunit Tubulin βPMCA Hoechst Merge Apoptotic Secondary necrosis Apoptotic + COL Apoptotic + COL +Z-vad Control +COL Control AMN PMCA AMN depolymerization PMCA disappears Apoptotic Apoptotic + COL Apoptotic + COL + z-VAD AMN depolymerization PMCA 0 10 20 30 40 50 60 70 80 90 100 PMCA + PMCA - Control Control +COL Apoptotic Apoptotic + COL Apoptotic +COL +Z-VAD Secondary Necrosis Percentage (%) ** a * a b * a c * a * a b * a c ab c Figure 4 PMCA-4 integrity during the execution phase of apoptosis and after AMN disorganization. (a) Fluorescence microscopy of PMCA-4 control cells, control cells treated with 2 mMcolchicine (Control þCOL) for 1 h, apoptotic cells, apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mMz-VAD (Apoptotic þCOL þz-VAD) for 1 h and secondary necrotic cells. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment and cytospined on a slide. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Then, cells were fixed and immunostained with anti-PMCA-4 (green) and anti-b-tubulin (red). Nuclear morphology was revealed by staining with Hoechst 33342. Bar ¼15 mm. (b) Magnification of apoptotic cells and apoptotic cells after 1 h of treatment with colchicine or colchicine plus z-VAD. (c) PMCA positive and negative cells were quantified by microscopic counting. More than 100 cells were examined for each experimental condition. *Po0.01 significant differences with respect to Control cells. a Po0.01, significant differences with respect to Apoptotic cells. b Po0.01, significant differences with respect to Apoptotic þCOL. c Po0.01, significant differences with respect to Apoptotic þCOL þz-VAD treated cells AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 6 Cell Death and Disease binactivation and subsequent sodium overload typical features of cells undergoing secondary necrosis. In previous works, we demonstrated that AMN during apoptosis depends on energized mitochondria. 14 As expected, in apoptotic cells with AMN, we found hyperpolarized mitochondria and high ATP levels (Figures 6c and d). On the contrary, when AMN was disorganized by colchicine o in secondary necrotic cells we found mitochondrial depolarization and low ATP levels (Figures 6c and d). Mitochondria depolarization and low ATP levels were prevented significantly in apoptotic cells without AMN by colchicine treatment when caspases were inhibited by z-VAD, suggesting that caspase-mediated cleavage of plasma membrane and cellular cortex proteins in the absent of AMN was important for inducing the bioenergetic collapse in apoptotic cells. High phosphatidylserine (PS) exposure in apoptotic cells with AMN. Phosphatidylserine (PS) is actively localized on the inner leaflet of the plasma membrane in healthy cells. The asymmetry of its distribution is lost in apoptotic cells. To examine the role of AMN in PS exposure, we analyzed the binding of annexin V-FITC to PS by immunofluorescence and flow cytometry analysis in control, apoptotic and secondary necrotic cells. Apoptotic cells were also treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD for 1 h to examine the effect of AMN depolymerization when caspases were active or inhibited. Figures 7a, b and c show clearly that PS was highly translocated in apoptotic cells with AMN. However, PS translocation was significantly reduced in apoptotic cells without AMN by colchicine treatment or cells in secondary Attached cells Floating cells Area (μm2)Perimeter (μm) 0 200 400 600 800 1,000 1,200 Cell area and perimeter * * a * a * a * a 0 10 20 30 40 50 60 70 80 90 100 a b Control Control +COL Apoptotic Apoptotic + COL Apoptotic +COL +Z-VAD Secondary Necrosis Control Control +COL Apoptotic Apoptotic + COL Apoptotic +COL +Z-VAD Secondary Necrosis Percentage (%) * a * a * a b * a b * a b c * a b c * a * a * a * a Figure 5 Effect of AMN disorganization in cell area and substrate detachment. (a) Cell area at the maximum cell perimeter was determined by microscopic studies using the Image J software in control cells, control cells treated with 2 mMcolchicine (Control þCOL), apoptotic cells, apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mMz-VAD (Apoptotic þCOL þz-VAD) for 1 h and secondary necrotic cells. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment and cytospined on a slide. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. More than 100 cells were examined for each experimental condition. (b) Floating and attached cells were quantified by microscopic counting. Data represent the means±S.D. of three independent experiments. *Po0.01 significant differences with respect to Control cells. a Po0.01, significant differences with respect to apoptotic cells. b Po0.01, significant differences with respect to Apoptotic þCOL. c Po0.01, significant differences with respect to Apoptotic þCOL þz-VAD treated cells AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 7 Cell Death and Disease necrosis. High levels of PS externalization were significantly recovered in apoptotic cells without AMN by colchicine treatment when caspases were inhibited by z-VAD, suggesting that caspase-mediated cleavage of plasma membrane and cellular cortex proteins also impairs PS externalization. These results suggest that proper PS externalization also depends on the presence of AMN. AMN in apoptotic cells enhances interactions with macrophages. As the presence of AMN in apoptotic cells effectively increase cell surface area and allows high PS externalization, we tested their influence upon binding and uptake by phagocytes. In the absence of AMN by colchicine treatment or in secondary necrotic cells, that is associated with low PS externalization, the proportion of macrophages interacting with and engulfing apoptotic cells was markedly reduced compared with apoptotic cells with AMN and high PS externalization (Figures 8a and b). However, the proportion of macrophages interaction/engulfing with apoptotic cells was restored in apoptotic cells without AMN by colchicine treatment when caspases were inhibited by z-VAD that also was associated with high PS externalization. Discussion In the present study, we show new data demonstrating that AMN indeed works as physical barrier impeding active caspases to reach and cleave critical proteins in the cellular LDH release ( %) 0 20 40 60 80 100 a cd b * * a b c * a * b ATP (nmoles/million cells) * 0 5 10 15 20 25 30 e * a b c * a * b Sodium levels (arbitrary units) Apoptotic +COL +Z-VAD Secondary Necrosis Control Control +COL Apoptotic Apoptotic +COL Secondary Necrosis Apoptotic +COL +Z-VAD 0 50 100 150 200 250 300 350 400 Control Control +COL Apoptotic Apoptotic +COL Calcium levels (arbitrary units) 0 20 40 60 80 100 120 140 Control Control +COL Apoptotic Apoptotic +COL Apoptotic +COL +Z-VAD Secondary Necrosis * * a b c * a * b Δψm (arbitrary units) * * a c * a * b 0 20 40 60 80 100 120 140 Apoptotic +COL +Z-VAD Secondary Necrosis Control Control +COL Apoptotic Apoptotic +COL Apoptotic +COL +Z-VAD Secondary Necrosis Control Control +COL Apoptotic Apoptotic +COL * * a * b * a b c Figure 6 Effect of microtubule disorganization on LDH release, intracellular calcium and sodium levels, mitochondrial membrane potential and ATP levels in apoptotic cells. LDH release, calcium and sodium levels, mitochondrial membrane potential and ATP levels were measured in control cells, control cells treated with 2 mMcolchicine (Control þCOL), apoptotic cells with AMN (Apoptotic), apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mM z-VAD (Apoptotic þ COL þz-VAD) for 1 h and secondary necrotic cells. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Material and Methods. Apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after the treatment. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. (a) After treatments, LDH release in fresh medium for 1 h was measured as is described in Material and Methods. (b) Intracellular calcium levels were measured by flow cytometry using the calcium indicator FLUO-4 (1 mM). (c) Intracellular sodium levels were measured by flow cytometry using the sodium indicator SBFI-AM (5 mM). (d) Mitochondrial membrane potential (DCm) was determined by MitoTracker Red staining and flow cytometry analysis as is described in Material and Methods. (e) ATP levels were determined by luminescence as is described in Material and Methods. *Po0.01 significant differences with respect to Control cells. a Po0.01, significant differences with respect to Apoptotic cells. b Po0.01, significant differences with respect to Apoptotic þCOL. c Po0.01, significant differences with respect to Apoptotic þCOL þz-VAD treated cells AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 8 Cell Death and Disease cortex and plasma membrane proteins to preserve plasma membrane integrity. We found that the AMN was present in all genuinely apoptotic cells but was disrupted in cells undergoing secondary necrosis. Furthermore, AMN disorganization in apoptotic cells by a short incubation with colchicine allowed caspase-mediated cleavage of cell cortex and plasma Secondary necrosis Apoptotic + COL Apoptotic + COL +Z-vad Control Tubulin βAnnexin V Hoechst Merge Apoptotic Apoptotic + COL Apoptotic + COL +z-VAD AMN AMN depolymerization AMN depolymerization Control +COL Apoptotic PS 0 100 200 300 400 500 600 Control Control +COL Apoptotic Apoptotic +COL Apoptotic +COL +Z-VAD Secondary Necrosis * * a c * a * b PS PS ab c Annexin V signal (arbitrary units) Figure 7 Effect of apoptotic microtubules on PS externalization. (a) Fluorescence microscopy of microtubules and PS externalization in control cells, control cell treated with colchicine for 1 h (Control þCOL), apoptotic cells with AMN (Apoptotic), apoptotic cells treated with 2 mMcolchicine (Apoptotic þCOL) for 1 h or colchicine plus 50 mM z-VAD (Apoptotic þCOL þz-VAD) for 1 h and secondary necrotic cells. H460 cells were grown on glass coverslips and apoptosis was induced as is described in the enriched apoptotic assay in Materials and Methods. Secondary necrotic cells were collected from the floating cells in de medium after treatment and cytospined onto a slide. Apoptotic cells were treated with colchicine or colchicine plus the pan-caspase inhibitor z-VAD to examine the effect of AMN depolymerization when caspases were active or inhibited. Then, cells were fixed and immunostained with anti-b-tubulin (red), and PS externalization was revealed by staining with Annexin V-FITC. Nuclear morphology was revealed by staining with Hoechst 33342 (1 mg/ml). Bar ¼15 mm). (b) Magnification of apoptotic cells and apoptotic cells after 1 h of treatment with colchicine or colchicine plus z-VAD. (c) PS externalization was quantified by flow cytometry as described in Material and Methods. Data represent the means±S.D. of three independent experiments. *Po0.01 significant differences with respect to Control cells. a Po0.01, significant differences with respect to Apoptotic cells. b Po0.01, significant differences with respect to Apoptotic þCOL. c Po0.01, significant differences with respect to Apoptotic þCOL þz-VAD treated cells AMN and free active caspase area in apoptosis M Oropesa-A ´vila et al 9 Cell Death and Disease