Ultraviolet Radiation Effects on Ehrlich Ascites Tumor Cells : Observations Using a Flying Spot Ultraviolet Microscope

A flying spot ultraviolet microscope, employing a fast scan and pulsed operation of the raster, has been used to induce radiation damage in ascites tumor slide cultures, and to study by time-lapse cinematography the progressive stages of cell damage. The cells observed came from a strain (EF₇) of the Ehrlich ascites carcinoma. Irradiated cells were found to show a characteristic syndrome of damage, involving blebbing at the cell surface, while control cells in the adjacent areas of the preparation remained unchanged. The end of the blebbing period is marked by swelling of the cells, and the time taken for this phenomenon to occur was used as a measure of the severity of the damage. It was found that the time required for swelling is dependent on the size of the dose employed, as well as on the sensitivity of the cells. This latter sensitivity was found to decline as the physiological age of the tumor increased. If ultraviolet illumination below 255 mµ is excluded, no symptoms of damage occur, even when very large doses are used. These observations are discussed in relation to the nature of the system in the cell which is affected.     

Ultraviolet radiation effects on Ehrlich ascites tumor cells; observations using a flying spot ultraviolet microscope

A flying spot ultraviolet microscope, employing a fast scan and pulsed operation of the raster, has been used to induce radiation damage in ascites tumor slide cultures, and to study by time-lapse cinematography the progressive stages of cell damage. The cells observed came from a strain (EF(7)) of the Ehrlich ascites carcinoma. Irradiated cells were found to show a characteristic syndrome of damage, involving blebbing at the cell surface, while control cells in the adjacent areas of the preparation remained unchanged. The end of the blebbing period is marked by swelling of the cells, and the time taken for this phenomenon to occur was used as a measure of the severity of the damage. It was found that the time required for swelling is dependent on the size of the dose employed, as well as on the sensitivity of the cells. This latter sensitivity was found to decline as the physiological age of the tumor increased. If ultraviolet illumination below 255 mmicro is excluded, no symptoms of damage occur, even when very large doses are used. These observations are discussed in relation to the nature of the system in the cell which is affected.     

Cell Blebbing and Membrane Area Homeostasis in Spreading and Retracting Cells

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.     

Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin

The cyclic heptapeptide hepatotoxin microcystin-LR from the cyanobacterium Microcystis aeruginosa induces rapid and characteristic deformation of isolated rat hepatocytes. We investigated the mechanism(s) responsible for cell shape changes (blebbing). Our results show that the onset of blebbing was accompanied neither by alteration in intracellular thiol and Ca2+ homeostasis nor by ATP depletion. The irreversible effects were insensitive to protease and phospholipase inhibitors and also to thiol-reducing agents, excluding the involvement of enhanced proteolysis, phospholipid hydrolysis, and thiol modification in microcystin-induced blebbing. In contrast, the cell shape changes were associated with a remarkable reorganization of microfilaments as visualized both by electron microscopy and by fluorescent staining of actin with rhodamine-conjugated phalloidin. The morphological effects and the microfilament reorganization were specific for microcystin-LR and could not be induced by the microfilament-modifying drugs cytochalasin D or phalloidin. Using inhibition of deoxyribonuclease I as an assay for monomeric actin, we found that the microcystin-induced reorganization of hepatocyte microfilaments was not due to actin polymerization. On the basis of the rapid microfilament reorganization and the specificity of the effects, it is suggested that microcystin-LR constitutes a novel microfilament-perturbing drug with features that are clearly different from those of cytochalasin D and phalloidin.     

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time‐lapse study

Background information. The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. Results. The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time‐lapse confocal microscopy. Fluorescently labelled proteins were not mis‐localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. Conclusion. These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.     

Cell shape and organelle modification in apoptotic U937 cells

U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode) and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis) were simultaneously analyzed.     

A Model of Blebbing in Mitotic Tissue Culture Cells

A preliminary study of blebbing in tissue cultures has been made. The tubal epithelium of fetal mouse oviduct was cultured at 37°C in Rose chambers. A cinematographic record was obtained of phase microscope observations of mitotic cells. Measurements of the size of both cells and blebs were made on the film using a “traveling” microscope. The duration and the rise and decay times of blebs were determined simply by counting frames on the film. Detailed observations are reported on blebbing in four cells undergoing mitosis. The results indicate that the frequency of blebbing as well as the duration of individual blebs exhibits a maximum during telophase. A model is proposed to account for blebbing in mitotic cells. The model attributes to local regions of the cell membrane the property of constant tension independent of stretch over some restricted range of stretch. This property implies that the cell membrane is locally unstable. Further assumptions stated explicitly in the model are that (i) cell division occurs at constant volume, (ii) the cell membrane stretches during cleavage, (iii) there is a positive pressure drop across the cell membrane. Evidence is cited in support of these assumptions as well as independent evidence that the cell membrane may be locally unstable. A physical model is described which would be expected to exhibit blebbing given the above assumptions.     

Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 microM microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.     

P2X7 nucleotide receptors mediate blebbing in osteoblasts through a pathway involving lysophosphatidic acid

Extracellular nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 receptors in many cell types, including osteoblasts. P2X7 receptors are ATP-gated cation channels that can induce formation of large membrane pores. Disruption of the gene encoding the P2X7 receptor leads to decreased periosteal bone formation and insensitivity of the skeleton to mechanical stimulation. Our purpose was to investigate signaling pathways coupled to P2X7 activation in osteoblasts. Live cell imaging showed that ATP or 2 ',3 '-O-(4-benzoylbenzoyl)-ATP (BzATP), but not UTP, UDP, or 2-methylthio-ADP, induced dynamic membrane blebbing in calvarial osteoblasts. Blebbing was observed in calvarial cells from wildtype but not P2X7 knock-out mice. P2X7 receptors coupled to activation of phospholipase D and A2, inhibition of which suppressed BzATP-induced blebbing. Activation of these phospholipases leads to production of lysophosphatidic acid (LPA). LPA caused dynamic blebbing in osteoblasts from both wild-type and P2X7 knock-out mice, similar to that induced by BzATP in wildtype cells. However, LPA-induced blebbing was more rapid in onset and was not affected by inhibition of phospholipase D or A2. Blockade or desensitization of LPA receptors suppressed blebbing in response to LPA and BzATP, without affecting P2X7-stimulated pore formation. Thus, LPA functions downstream of P2X7 receptors to induce membrane blebbing. Furthermore, inhibition of Rho-associated kinase abolished blebbing induced by both BzATP and LPA. In summary, we propose a novel signaling axis that links P2X7 receptors through phospholipases to production of LPA and activation of Rho-associated kinase. This pathway may contribute to P2X7-stimulated osteogenesis during skeletal development and mechanotransduction.     

Development of Apical Blebbing in the Boar Epididymis

Microvesicles are of increasing interest in biology as part of normal function of numerous systems; from the immune system (T cell activation) to implantation of the embryo (invasion of the trophoblasts) and sperm maturation (protein transfer in the epididymis). Yet, the mechanisms involved in the appearance of apical blebbing from healthy cells as part of their normal function remain understudied. Microvesicles are produced via one of two pathways: exocytosis or apical blebbing also termed ectocytosis. This work quantifies the histological appearance of apical blebbing in the porcine epididymis during development and examines the role of endogenous estrogens in regulating this blebbing. Apical blebbing appears at puberty and increases in a linear manner into sexual maturity suggesting that this blebbing is a mature phenotype. Endogenous estrogen levels were reduced with an aromatase inhibitor but such a reduction did not affect apical blebbing in treated animals compared with their vehicle-treated littermates. Epididymal production of apical blebs is a secretion mechanism of functionally mature principal cells regulated by factors other than estradiol.     

Marginal blebbing during the early stages of TNF-induced apoptosis indicates alteration in actomyosin contractility

Dynamics of alterations of cell surface topography during TNF-induced apoptosis of HeLa cells was examined by phase-contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4-6 h of incubation but much earlier, after 1.5-3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1-2 h. This phenomenon was called "marginal blebbing". It took place before the cytochrome c release from mitochondria to cytosol. Marginal blebbing was inhibited by drugs that depolymerized actin microfilaments (cytochalasin, latrunculin) or decreased Rho-kinase-dependent contractility of actin-myosin cortex (H7, HA-1077, Y27632). A pancaspase inhibitor, zVAD-fmk, completely prevented marginal and general blebbing, and TNF-induced apoptosis. DEVD-fmk, a specific inhibitor of caspase-3, inhibited both marginal and general blebbing but not cell rounding and death. Thus, marginal blebbing is an early microfilament-dependent apoptotic event. It is suggested that it is initiated by minimal activation of caspase-3 and the following local Rho-kinase-dependent stimulation of actin-myosin cortex contractility. Localization of marginal blebs at the active edge may be associated with special organization of cortex in that zone.     

Response in shape and size of individual p31 cancer cells to cisplatin and ouabain: a computerized image analysis of cell halo characteristics during continuous perfusion

BACKGROUND: Volume regulation is essential for cellular functions, including cell death, such as apoptosis. Flow cytometry is standard for nonadherent cells, such as blood cells. Our aim was to explore image analysis methods to study adherent cancer cells of a solid tumor. METHODS: P31 mesothelioma cells were perifused (40 min) and studied by phase-contrast microscopy. A noise reduction of the cell contour was tested to more accurately yield the cell shape factor (SF). The optical halo around the cell was analyzed for information about membrane blebbing. RESULTS: The projected cell area (PCA) slowly increased under control perfusion, the halo outside more than the halo inside. Cisplatin (apoptosis) caused an immediate increase in the PCA-halo outside (5.9 +/- 1.2 %, P < 0.01, 1-5 min) and the SF indicated decreased roundness (P < 0.05). The SF-halo inside became more irregular than the outside, which was different from the control cells. The morphology reflected instant blebbing, and the cell bodies showed fragmentation after about 20 min. Ouabain resulted in only small changes in PCA and SF, significantly different from both control and cisplatin conditions. CONCLUSIONS: Image analysis (PCA and SF) on perifused adherent cancer cells may serve as a tool to follow the sensitivity of cancer chemotherapy and to study cell death patterns.     

T-lymphocyte mediated lysis of tumor cells in the presence of alloantiserum.

The blocking effects of tumor alloantiserum (AS) on the process of T-lymphocyte mediated cytolysis of target cells was investigated with the scanning electron microscope by analyzing the frequency of lymphocyte-target cell interactions and the respective changes in target cell morphology at various time intervals of the cytolytic process. Our results demonstrate that in the presence of AS the frequencies of lymphocyte-target cell conjugates in which the target cells were undergoing active blebbing correlated with the delayed kinetics of ⁵¹Cr release. Our data also show that the AS did not interfere significantly with the binding of lymphocytes to target cells, but delayed the appearance of surface blebbing of the target cells. Thus, effector lymphocytes required a prolonged time of continuous interaction with the target cells in order to exert their cytolytic effects in the presence of AS. This conclusion was further confirmed by experiments in which lymphocyte-target cell interactions were interrupted by the addition of EDTA to the culture medium.     

Microtubules suppress blebbing and stimulate lamella extension in spreading fibroblasts

We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment, cells start blebbing, which continues for different times and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic way through stochastic formation of lamellipodia. A second mode that occurs in 15% of cells was rapid, isotropic spreading via formation of circular lamella. The rate of spreading was maximal at the beginning and decreased during the first hour according to a logarithmic law. After 60 min, many cells formed stable edges and started to migrate on the substrate. However, the cell area slowly continued to increase. Actin bundles were formed 20 min after cell attachment. They first run along the cell boundary. This system disassembled within 20–40 min and was substituted with stress fibers crossing the cell. In isotropically spread cells, no actin bundles were seen. Microtubules in the spreading cells enter into large blebs and all nascent lamellas; later, they form a radial array. When MTs have been depolymerized or stabilized blebbing started, before cells attach to the substrate and continue much longer than in control cells. After both treatments, the initial rate of spreading decreases several-fold and remains constant for many hours. After 24 h, the mean area occupied by cells with an altered MT system was the same as in control. Alteration of the MT system had a moderate effect on the actin system: formation of actin cables occurred at the same time as in control (within 20 min upon cell attachment); however, they started growing even in cells undergoing prolonged blebbing. Actin cables running along the cell margin were similar to those in control cells, but they did not disappear for up to 1 h. When stabilized, MTs form a chaotic array: they do not enter blebs and, in spread cells, run parallel to the cell margin at a distance of 3–5 μm. We conclude that dynamic MTs speed up completion of blebbing and promote early stages of fibroblast spreading.     

DNA degradation and changes in the permeability and form of Ehrlich ascites tumor cells when incubated in an anaerobic medium without glucose

After a 3-hour incubation of the Ehrlich ascite tumor cells in buffered Hanks solution, without glucose and oxygen, the extensive cell injuries were observed. The time-course of appearance of these injuries was as follows: cell blebbing, staining of the cells with trypan blue, and then their staining with ethidium bromide. The DNA degradation registered with fluorometric method coincided in time with cell staining with trypan blue. All injuries (except DNA degradation) were delayed at pH 6.0 compared with those at pH 7.3. Glucose added to the cell suspension greatly protected the cells from these injuries, although DNA degradation at pH 6.0 in these conditions was a little higher than that at pH 7.3.     

MgcRacGAP regulates cortical activity through RhoA during cytokinesis

Although Rho GTPases regulate multiple cellular events, their role in cell division is still obscure. Here we show that expression of a GTPase-activating protein (GAP)-deficient mutant (R386A) of the Rho regulator MgcRacGAP induces abnormal cortical activity during cytokinesis in U2OS cells. Multiple large blebs were observed in cells expressing MgcRacGAP R386A from the onset of anaphase to the late stage of cell division. When mitotic blebbing was excessive, cytokinesis was inhibited, and cells with micronuclei were generated. It has been reported that blebbing is caused by abnormal cortical activity. The MgcRacGAP R386A-induced abnormal cortical activity was inhibited by the dominant negative form of RhoA, but not Rac1 or Cdc42. Moreover, expression of constitutively active RhoA also induced drastic cortical activity during cytokinesis. Unlike apoptotic blebbing, MgcRacGAP R386A-induced blebbing was not inhibited by the ROCK inhibitor Y-27632, suggesting that MgcRacGAP regulates cortical activity during cytokinesis through a novel signaling pathway. We propose that MgcRacGAP plays a pivotal role in cytokinesis by regulating cortical movement through RhoA.     

Membrane blebbing is associated with Ca2+-activated hyperpolarizations induced by serum and alpha 2-macroglobulin

We have reported previously that serum and alpha 2-macroglobulin (alpha 2M) induce Ca2+-activated hyperpolarizations in the membrane potential of a clonal rat osteosarcoma cell line (ROS 17/2) (Dixon and Aubin, J. Cell, Physiol., 132:215-225, 1987). In this report, we describe morphological changes that accompany these hyperpolarizations. Both cell surface blebbing (zeiosis) and transient hyperpolarizations were induced by application of 10% fetal bovine serum (FBS) or alpha 2M; neither was induced by serum-free medium, a suspension of latex beads, or purified bovine serum albumin. Following a brief application of FBS or alpha 2M at time 0, electrical activity typically occurred between 7-40 s and was always followed by blebbing activity that began at 30 s and persisted for 3-5 min. In contrast, continuous exposure to FBS resulted in the persistence of both blebbing activity and transient hyperpolarizations for periods of at least several hours. Scanning electron microscopy (SEM) revealed that the blebs appeared concomitantly with the disappearance of microvilli and the appearance of surface pits that measured 100-300 nm in diameter. Coated pits and vesicles, similar in size to the pits observed by SEM, were observed using transmission electron microscopy (TEM). By TEM, blebs were found to contain few organelles other than centrally located free ribosomes. Fluorescence microscopy of nitrobenzooxadizole-phallacidin-labeled cells indicated that blebs contained filamentous actin and that microfilament bundles remained primarily on the substratum side of blebbed cells. We propose that blebbing results from a dynamic local reorganization of microfilaments initiated by ligand-induced transient increases in intracellular Ca2+.     

KIF13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.     

Interaction and fusion dynamics between cellular blebs

Membrane blebbing, as a mechanism for cells to regulate their internal pressure and membrane tension, is believed to play important roles in processes such as cell migration, spreading and apoptosis. However, the fundamental question of how different blebs interact with each other during their life cycles remains largely unclear. Here, we report a combined theoretical and experimental investigation to examine how the growth and retraction of a cellular bleb are influenced by neighboring blebs as well as the fusion dynamics between them. Specifically, a boundary integral model was developed to describe the shape evolution of cell membrane during the blebbing/retracting process. We showed that a drop in the intracellular pressure will be induced by the formation of a bleb whose retraction then restores the pressure level. Consequently, the volume that a second bleb can reach was predicted to heavily depend on its initial weakened size and the time lag with respect to the first bleb, all in quantitative agreement with our experimental observations. In addition, it was found that as the strength of membrane-cortex adhesion increases, the possible coalescence of two neighboring blebs changes from smooth fusion to abrupt coalescence and eventually to no fusion at all. Phase diagrams summarizing the dependence of such transition on key physical factors, such as the intracellular pressure and bleb separation, were also obtained.     

Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.