Depolarization of the membrane potential by hyaluronan

The membrane potential is mainly maintained by the K⁺ concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high‐molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration‐dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high‐molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high‐molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high‐molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism. J. Cell. Biochem. 111: 858–864, 2010. © 2010 Wiley‐Liss, Inc.     

Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling-induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Age-related changes in hyaluronate uptake by cultured fibroblasts

Skin fibroblasts from chick embryos of 7 and 14 days incubation have been tested for their ability to bind, internalize and degrade [3H]-hyaluronan. Seven-day fibroblasts internalize and degrade a greater amount of labelled hyaluronan than 14-day cells. The implications of these findings in the regulation by the fibroblasts of extracellular matrix composition in glycosaminoglycans are discussed.     

Reduced supportive capacity of bone marrow stroma upon chemotherapy is mediated via changes in glycosaminoglycan profile.

High dose chemotherapy and radiation have been found to impair the hematopoiesis-supportive capacity of bone marrow stroma. We now provide evidence for an important role of chemotherapy-induced alterations in stromal glycosaminoglycans (GAGs) in reduction of the supportive properties of stromal fibroblasts. Exposure to cytarabine resulted in a pronounced increase in hyaluronan, both in the cell/matrix (p<0.03) and supernatant fraction (p<0.05). Gene expression analysis showed a corresponding increase in gene expression of hyaluronan synthase 1, indicating that the increase in hyaluronan is at least partly under genetic control. Functionally, hyaluronan significantly inhibited the proliferation of early megakaryocytic progenitor cells in a dose dependent way (p=0.01). The increase in hyaluronan was confirmed in vivo by showing a >2-fold increase in bone marrow hyaluronan of patients after chemo- and/or radiotherapy as conditioning for an allogeneic stem cell transplantation, indicating physiologically relevance. Furthermore, there was a trend towards a decrease in the amount and sulfation of stromal heparan sulfate proteoglycans upon exposure to cytarabine, resulting in a 40% reduced binding of SDF1-alpha to stromal cells (p<0.05). In conclusion, there is a pronounced effect of cytarabine treatment on the expression of genes involved in GAG synthesis and degradation, affecting the synthesis and function of stromal GAGs. Our results indicate that chemotherapy-induced changes in stromal GAG profile are likely to affect normal hematopoiesis.     

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.     

Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.     

Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.     

TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.     

Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth.     

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.     

nsPEF-induced PIP2 depletion,PLC activity and actin cytoskeletal cortex remodeling are responsible for post-exposure cellular swelling and blebbing

Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP₂) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP₂ is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP₂ depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting “permeabilization” of the PM.     

MDA-MB-231 breast cancer cell viability,motility and matrix adhesion are regulated by a complex interplay of heparan sulfate,chondroitin−/dermatan sulfate and hyaluronan biosynthesis

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.     

Modulation of KCNQ4 channel activity by changes in cell volume

KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling.     

Prostaglandin induced shape changes in fibroblasts grown in cell culture

PGE2 induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE2 at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hours. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE2 resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF2 alpha did not however induce similar changes. This difference between PGE2 and PGF2 alpha is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE2 may have the ability to induce shape changes in cells.     

Drosophila grim induces apoptosis in mammalian cells.

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.     

Pendrin overexpression affects cell volume recovery, intracellular pH and chloride concentration after hypotonicity-induced cell swelling

The pendrin (SLC26A4 or PDS) gene is responsible, when mutated, for the Pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid dysfunctions. Pendrin protein is an anion exchanger and we focused on a still unexplored function that it might play in view of its importance in the inner ear: Cl(-) fluxes regulation during cellular volume control. We challenged HEK-293 Phoenix cells over-expressing wild type pendrin (PDS HEK cells) together with the EYFP (Enhanced Yellow Fluorescent Protein) or over-expressing the EYFP alone (control HEK cells) with hypo-osmolar solutions. Taking advantage of the confocal optical sectioning we measured the cell volume. In addition, we determined the intracellular pH and chloride concentration with fluorescent probes (EYFP and seminaphthorhodafluor-5F, SNARF-5F). Consequently, we could estimate simultaneously Cl(-) fluxes, cellular volume and intracellular pH variations. Cl(-) movements markedly differed between PDS and control HEK cells upon hypotonic shock and are accompanied by an attenuation of the swelling induced pH drop in PDS HEK cells. The contemporary measurements of the three variables not yet reported in living cells, allowed to assess a possible influence of pendrin upregulation in volume homeostasis and evidenced its participation to Cl(-) fluxes.     

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+.     

Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages

Chronic inhalation of crystalline silica is an occupational hazard that results in silicosis due to the toxicity of silica particles to lung cells. Alveolar macrophages play an important role in clearance of these particles, and exposure of macrophages to silica particles causes cell death and induction of markers of apoptosis. Using time-lapse imaging of MH-S alveolar macrophages, a temporal sequence was established for key molecular events mediating cell death. The results demonstrate that 80 % of macrophages die by apoptosis and 20 % by necrosis by clearly distinguishable pathways. The earliest detectable cellular event is phago-lysosomal leakage, which occurs between 30 and 120 min after particle uptake in both modes of death. Between 3 and 6 h later, cells undergoing apoptosis showed a dramatic increase in mitochondrial transmembrane potential, closely correlated with activation of both caspase-3 and 9 and cell blebbing. Externalization of phosphatidyl serine and nuclear condensation occurred 30 min–2 h after the initiation of cell blebbing. Cells undergoing necrosis demonstrated mitochondrial membrane depolarization but not hyperpolarization and no caspase activation. Cell swelling followed the decrease in mitochondrial membrane potential, distinguishing necrosis from apoptosis. All cells undergoing apoptosis followed the same temporal sequence, but the time lag between phago-lysosomal leakage and the other events was highly variable from cell to cell. These results demonstrate that crystalline silica exposure can result in either apoptosis or necrosis and each occurs in a well-defined but temporally variable order. The long time gap between phago-lysosomal leakage and hyperpolarization is not consistent with a simple scenario of phago-lysosomal leakage leading directly to cell death. The results highlight the importance of using a cell by cell time-lapse analysis to investigate a complex pathway such as silica induced cell death.