Blebbing confers resistance against cell lysis.

The plasma membrane constitutes a barrier that maintains the essential differences between the cytosol and the extracellular environment. Plasmalemmal injury is a common event during the life of many cells that often leads to their premature, necrotic death. Blebbing - a display of plasmalemmal protrusions - is a characteristic feature of injured cells. In this study, we disclose a previously unknown role for blebbing in furnishing resistance to plasmalemmal injury. Blebs serve as precursors for injury-induced intracellular compartments that trap damaged segments of the plasma membrane. Hence, loss of cytosol and the detrimental influx of extracellular constituents are confined to blebs that are sealed off from the cell body by plugs of annexin A1 - a Ca(2+)- and membrane-binding protein. Our findings shed light on a fundamental process that contributes to the survival of injured cells. By targeting annexin A1/blebbing, new therapeutic approaches could be developed to avert the necrotic loss of cells in a variety of human pathologies.

Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells.

Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca(2+) concentration [Ca(2+)](i) below a critical threshold of 10 microM, repair mechanisms seal off 'hot spots' of Ca(2+) entry and shed them in the form of microparticles, leading to [Ca(2+)](i) reduction and cell recovery. Cells that are capable of preventing an elevation of [Ca(2+)](i) above the critical concentration, yet are unable to complete plasma membrane repair, enter a prolonged phase of [Ca(2+)](i) oscillations, accompanied by a continuous shedding of microparticles. When [Ca(2+)](i) exceeds the critical concentration, an irreversible formation of ceramide platforms within the plasma membrane and their internalisation drives the dying cells beyond the 'point of no return'. These findings show that the extent of [Ca(2+)](i) elevation determines the fate of targeted cells and establishes how different Ca(2+)-dependent mechanisms facilitate either cell survival or death.

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia.

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.

Regulation of ecto-5'-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?

The spatial segregation of the plasma membrane plays a prominent role in distinguishing and sorting a large number of signals a cell receives simultaneously. The plasma membrane comprises regions known as lipid rafts, which serve as signal-transduction hubs and platforms for sorting membrane-associated proteins. Ca(2+)-binding proteins of the annexin family have been ascribed a role in the regulation of raft dynamics. Glycosylphosphatidylinositol-anchored 5'-nucleotidase is an extracellular, raft-associated enzyme responsible for conversion of extracellular ATP into adenosine. Our results point to a regulation of ecto-5'-nucleotidase activity by Ca(2+)-dependent, annexin-mediated stabilization of membrane rafts.

Increased cholesterol decreases uterine activity: functional effects of cholesterol alteration in pregnant rat myometrium.

Uterine quiescence is essential for successful pregnancy. Cholesterol and triglycerides are markedly increased in pregnancy. Cholesterol is enriched in microdomains of the plasma membrane known as rafts and caveolae. Both lipid rafts and caveolae have been implicated in cellular signaling cascades. The purpose of this work was to investigate whether manipulation of cholesterol content alters uterine contractility. Late pregnancy (19-21 days) rats were humanely euthanized and strips of longitudinal myometrium were then dissected. Force and Ca(2+) measurements were simultaneously recorded and cholesterol increased by the addition of 5 mg/ml cholesterol or 0.25 mg/ml low-density lipoproteins (LDLs) or reduced by 2% methyl-beta-cyclodextrin (MCD) or 2 U/ml cholesterol oxidase addition to the perfusate. Both LDLs and cholesterol profoundly inhibited spontaneous uterine force production and associated Ca(2+) transients; frequency, amplitude, and duration of contraction were all significantly reduced compared with preceding control contractions. Force and Ca(2+) were also reduced by cholesterol when 1 nM oxytocin was used to stimulate the myometrium. Uterine activity was significantly increased by cholesterol extraction with MCD or cholesterol oxidase treatment. Electron microscopy confirmed the lipid raft disrupting effect of MCD, as formerly electron microscopy-visible caveolae in the myometrial cell membrane all but disappeared after MCD treatment. These data show that uterine smooth muscle cell cholesterol content is critically important for functional activity. A novel finding of our study is that cholesterol is inhibitory for force generation. It may be one of the mechanisms operating to maintain uterine quiescence throughout gestation and may also contribute to difficulties in labor suffered by obese women.

Membrane cholesterol regulates smooth muscle phasic contraction.

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts-and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K(+)-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its alpha-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertension.

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions.

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells.

Muscle contraction implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the annexin family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of annexins 2 and 6, and a significant decrease in raft-associated 5'-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties.

Stress fibres-a Ca2+ -independent store for annexins?

Annexins belong to a family of lipid-binding proteins that are implicated in membrane organization. Several members are capable of binding to actin and, in smooth muscle cells, annexin 6 is known to form a Ca(2+)-dependent, plasmalemmal complex with actin filaments. Annexins can also associate with F-actin containing stress fibres within cultured smooth muscle cells or fibroblasts in a Ca(2+)-independent manner. Depolymerization of stress-fibre systems with cytochalasin D leads to the translocation of actin-bound annexin 2 from the cytoplasm to the plasma membrane at high intracellular levels of Ca(2+). This type of Ca(2+)-dependent annexin mobility is observed only in cells of mesenchymal phenotype, which have a well-developed stress-fibre system; not in epithelial cells.

Annexins in cell membrane dynamics. Ca(2+)-regulated association of lipid microdomains.

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca(2+) concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca(2+) sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca(2+) concentrations. Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191-35195). Annexin II promotes the Ca(2+)-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca(2+) flux.

Smooth muscle actomyosin promotes Ca2+-dependent interactions between annexin VI and detergent-insoluble glycosphingolipid-enriched membrane domains.

The mechanical link coupling cytoskeletal and contractile proteins to the sarcolemma of smooth muscle cells is essential for transmitting tension from the cell's interior to exterior. In addition to the well-characterized actin-integrin associations present in adhaerens junctions, our recent work has postulated the existence of a reversible annexin-dependent membrane-cytoskeleton complex, forged in response to a rise in intracellular Ca2+ concentration following smooth muscle cell stimulation (Babiychuk et al., J. Biol Chem. 1999, 274, 35191-35195). Detailed biochemical characterization of the interactions responsible for the formation of this complex revealed that annexins II and VI interact with actomyosin, or detergent-insoluble glycosphingolipid-enriched membrane domains (rafts) purified from smooth muscle, in a concentration- and Ca2+-dependent manner. Annexin II interacted with lipid rafts with high Ca2+-sensitivity, while for annexin VI this interaction required non-physiologically high concentrations of free Ca2+. However, the Ca2+-sensitivity of the latter interaction strongly increased in the presence of purified smooth muscle actomyosin. The detailed biochemical analysis of the interactions occurring between annexin II, annexin VI, actomyosin and rafts suggests that annexins regulate sarcolemmal organization during smooth muscle cell contraction.

Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells.

The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction.

Oligomerization of smooth muscle myosin light chain kinase and its modifications by melittin and calmodulin.

The catalytic activity of smooth muscle myosin light chain kinase (MLCKase) requires the presence of calcium and calmodulin [CM; J. T. Stull et al. (1993) Molecular and Cellular Biochemistry, Vols. 127/128, pp. 229-237] and can also be modified through its own oligomerization [E. B. Babiychuk et al. (1995) Biochemistry, Vol. 34, pp. 6366-6372]. In the present report we demonstrate that melittin, one of the most potent CM antagonists, interacted reversibly with the MLCKase apoenzyme with affinities comparable to those of CM and influenced the oligomeric state of the kinase. At low melittin to kinase ratios the kinase formed insoluble oligomers (aggregates) while at higher melittin concentrations it existed predominantly as soluble oligomers revealed by cross-linking as octamers and hexamers. The kinase alone exhibited similar biphasic solubility within a 5-30 microM range and its solubility was strongly influenced by the ionic strength of the medium. Melittin was also found to promote both the aggregation of the purified 24-kDa C-terminal fragment of the kinase and its analogue telokin, as well as of myosin light chains, but had no effect on the solubility of bovine serum albumin, caldesmon, or calmodulin. These data and our cross-linkage experiments indicate that the insoluble kinase oligomers arose via melittin-induced aggregation of the kinase dimers in which the relokin-like domain played a main role. The soluble oligomers, in turn, were formed after saturation of the kinase with melittin, which resulted in a weakening of the interaction between the protomers with an increase of the long-range order within the oligomers. This interpretation is consistent with the observed effects of melittin on MLCKase catalytic and autocatalytic activities. At concentrations of melittin required to produce soluble oligomers, the binding of the kinase to myosin filaments was considerably enhanced. A plausible mechanism for the formation of the soluble oligomers and aggregates is suggested and its relation to the possible MLCKase assemblies discussed in terms of a model.

Purification and characterization of a kinase-associated, myofibrillar smooth muscle myosin light chain phosphatase possessing a calmodulin-targeting subunit.

A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase- and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca2+-independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10-20-fold after preincubation with 5-50 microM of CoCl2; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (Ki = 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 microM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.

Modulation of smooth muscle myosin light chain kinase activity by Ca2+/calmodulin-dependent, oligomeric-type modifications.

Oligomerization of turkey gizzard myosin light chain kinase (MLCKase) was demonstrated by a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a standard reagent used in investigations of specific protein-protein interaction [Mornet et al. (1989) J. Muscle Res. Cell Motil. 10, 10-24]. This approach revealed that in solution the kinase was not monomeric but the monomers were in equilibrium with the kinase dimeric and oligomeric forms. Addition of Ca2+/calmodulin (CM) shifted this equilibrium in the direction of the kinase dimers, accompanied by a 2-fold decrease of the kinase catalytic activity, in addition to a 2-fold decrease of its apparent affinity for CM [Sobieszek et al. (1993) Biochem. J. 295, 405-411]. The dimer (and/or oligomer) formation was shown to result from an interaction of the kinase autoinhibitory domain with its 24 kDa tryptic fragment containing titin-like domain II-3. The possible significance of the oligomerization in regulation of MLCKase activity is discussed.

Significance of the N-terminal fragment of myosin regulatory light chain for myosin-actin interaction.

The influence of myosin regulatory light chains (LC2s) lacking the 2kD N-terminal portions of these chains, on internal organization of myosin heads and on actin-myosin interaction was studied with limited proteolysis and polarized fluorescence methods. For these studies heavy meromyosin (HMM) preparations were used: HMM containing intact LC2s (phosphorylated or dephosphorylated) and HMM containing LC2s lacking the 2kD N-terminal portions (including serine which can be phosphorylated). It was found that the susceptibility of the heavy chain cleavage site to trypsin and the alkali light chain (LC1) site to papain of myosin containing shortened regulatory LC2s, is not dependent on saturation of LC2s with Ca2+ or Mg2+ ions. This is in contrast to the myosin containing intact LC2s where Ca2+ or Mg2+ ion saturation does demonstrate a dependence. Similarly, in spectroscopic experiments, dephosphorylated HMM containing intact LC2s causes decrease or increase of actin filament flexibility depending on whether Mg2+ or Ca2+ are bound to LC2s. Correspondingly, HMM with shortened LC2s induces only increase of actin flexibility despite cations being bound. We conclude that the N-terminal fragment of LC2 is important for ensuring a proper Ca2+ dependent conformation of myosin head in the course of its actin-activated ATP hydrolysis.

Skeletal muscle myosin regulatory light chains conformation affects the papain cleavage of A1 light chains.

In the present study, the influence of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (RLC) on accessibility of myosin and heavy meromyosin alkali light chains (A1) for papain digestion was investigated. The properties of native and papain treated myosin and heavy meromyosin were compared. Exchange of magnesium ions bound to RLCs for calcium ions accelerates the digestion of A1 in the presence of ATP in dephosphorylated myosin, heavy meromyosin, acto-myosin and the acto-heavy meromyosin complex. In the absence of ATP the exchange of magnesium ions bound to RLCs for calcium ions delays the digestion of A1 in the acto-myosin complex. Myosin and heavy meromyosin having shortened A1 by papain cleavage shows decreased K(+)-ATPase and increased actin binding ability in the presence and absence of ATP. The cooperation of RLC and A1 with heavy chains in the changes of structural organization of myosin head during muscle contraction is discussed.

Ca(2+)-calmodulin-dependent modification of smooth-muscle myosin light-chain kinase leading to its co-operative activation by calmodulin.

It has recently been shown that at relatively high molar ratios of myosin light-chain kinase (MLCKase) to calmodulin (CM) almost complete inhibition of the kinase activity occurs [Sobieszek (1991) J. Mol. Biol. 220, 947-957]. This inhibition resulted in a highly co-operative activation of MLCKase by CM, whereas the opposite activation (of CM by kinase) was hyperbolic, as expected (unco-operative). This difference in activation was observed only for kinase preparations preincubated with sub-stoichiometric amounts of CM, and only when micromolar concentrations of Ca2+ were present. The inhibitory effect was variable and depended not only on the concentration ratio of kinase to CM but also on the MLCKase preparation. For most of the preparations full inhibition required 5-15 min of preincubation at 25 degrees C and a 3-6-fold molar excess of kinase over CM. The inhibition was reversible, since full activity could be obtained after saturation of the kinase by additional CM. The inhibitory effect did not require ATP (excluding phosphorylation-type modifications of the kinase), and dephosphorylation of the kinase was not involved, since inhibition of an endogenous MLCK phosphatase by microcystin-LR did not decrease the inhibitory effect. Since the co-operative activation by CM was observed for cross-linked MLCKase preparations enriched in kinase dimers, but was absent for the analogous preparations enriched in the oligomers, we concluded that Ca(2+)-CM-dependent changes in the oligomeric state of the kinase were responsible for the modification observed. The exact nature of these modifications remains to be established.

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.