Calpain is required for the rapid, calcium-dependent repair of wounded plasma membrane

Mammalian cells require extracellular calcium ion to undergo rapid plasma membrane repair seconds after mechanical damage. Utilizing transformed fibroblasts from calpain small subunit knock-out (Capns1-/-) mouse embryos, we now show that the heterodimeric, typical subclass of calpains is required for calcium-mediated survival after plasma membrane damage caused by scraping a cell monolayer. Survival of scrape-damaged Capns1-/- cells was unaffected by calcium in the scraping medium, whereas more Capns1+/+ cells survived when calcium was present. Calcium-mediated survival was increased when Capns1-/- cells were scraped in the presence of purified m- or mu-calpain. Survival rates of scraped Capns1+/+, HFL-1, or Chinese hamster ovary cells were decreased by the calpain inhibitor, calpeptin, or the highly specific calpain inhibitor protein, calpastatin. Capns1-/- cells failed to reseal following laser-induced membrane disruption, demonstrating that their decreased survival after scraping resulted, at least in part, from failed membrane repair. Proteomic and immunologic analyses demonstrated that the known calpain substrates talin and vimentin were exposed at the cell surface and processed by calpain following cell scraping. Autoproteolytic activation of calpain at the scrape site was evident at the earliest time point analyzed and appeared to precede proteolysis of talin and vimentin. The results indicate that conventional calpains are required for calcium-facilitated survival after plasma membrane damage and may act by localized remodeling of the cortical cytoskeleton at the injury site.     

Seminal plasma proteins revert the cold-shock damage on ram sperm membrane

Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.     

Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry

Using electron microscopic immunocytochemistry, we have investigated the intracellular location of the src protein (p21) in cells transformed by the Harvey strain of Murine Sarcoma Virus (Ha-MSV). Antibodies to p21 were derived from tumor-bearing rats inoculated with Ha-NRK cells. The distribution of p21 in intracellular sites in MDCK dog cells transformed by Ha-MSV was examined and quantified using a recently developed immunocytochemical technique. More than 95% of p21 was localized to the inner surface of the plasma membrane in these Ha-MSV-transformed cells; p21 was not exposed on the outside surface of the plasma membrane. A similar location was observed by immunofluorescence in other Ha-MSV-transformed cell lines, including cells derived from rat, mouse and mink. This finding, and the previous demonstration that p60src of avian sarcoma virus is concentrated on the inner surface of the plasma membrane, suggests that the plasma membrane is a major site of action for transforming proteins.     

Tissue Distribution of ERp61 and Association of Its Increased Expression with IgG Production in Hybridoma Cells

A protein of molecular weight 60 kDa was purified from the culture medium of a murine colon carcinoma cell line, colon26, and its partial amino-acid sequence determined. Extremely high homology was found with the deduced sequence from cDNA of rat ERp61, earlier found to be an endoplasmic reticulum (ER)-resident protein with redox activity and a similar structure to protein disulfide isomerase (PDI). Western blotting analysis showed that colon26 cells secrete a significant amount of ERp61 into culture medium, although most remains intracellular. The thiol:protein disulfide oxidoreductase activity of the purified mouse ERp61 was demonstrated by insulin-reduction assay. The ER location of the protein in fibroblasts was immunocytochemically confirmed by double staining for ERp61 and another ER-resident protein, PDI or Hsp47. Immunohistochemical studies of murine tissues showed a ubiquitous distribution of ERp61 in a wide variety of cell types. However, it was particularly abundant in plasma cells, mucus-secreting cells in various tissues, neuroendocrine cells including neurons, and follicular epithelia of thyroid gland that actively synthesize and secrete proteins containing cysteine residues. Furthermore, a high correlation was observed between intracellular amounts of ERp61 and immunoglobulin production by hybridoma cells. These results indicate that ERp61 may be involved in disulfide bond formation for such proteins.     

Structure and assembly of the endoplasmic reticulum: biosynthesis and intracellular sorting of ERp61, ERp59, and ERp49, three protein components of murine endoplasmic reticulum

Rabbit antibodies have been prepared against ERp61, ERp59, and ERp49, three protein components of rough endoplasmic reticulum (RER) purified from mineral oil-induced plasmacytoma 315 (MOPC-315) tissue. Analysis of subcellular fractions of MOPC-315 tissue by an immunoprecipitation procedure demonstrated that all three endoplasmic reticulum proteins (ERps) were most enriched in the RER. Immunologically cross-reacting proteins of similar molecular weight have been detected in other eucaryotic cell lines. We have used these antibodies to study the post-translational processing and biosynthetic sorting of the three ERps in pulse-labeled MOPC-315 cells. No larger precursor forms of the ERps were detected and none of the ERps were found to possess asparagine-linked oligosaccharide moieties. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 cells to study the biosynthetic sorting of ERp61, ERp59 and ERp49 and have found no evidence to suggest that these proteins ever leave the endoplasmic reticulum. In addition, all three ERps appeared to have luminally exposed domains. ERp61 and ERp59 were entirely protected by the ER membrane in the absence of detergent, while ERp49 was a transmembrane protein that also possesses a cytoplasmically exposed domain. We have used the anti-ERp antibodies to quantitate the synthesis and accumulation of the three ERps during lipopolysaccharide (LPS)-induced lymphocyte differentiation. After 48 h of culture in the presence of LPS, the synthesis of ERp49 increased sixfold relative to that in control cells. The synthesis and membrane accumulation of ERp61 and ERp59 were less affected by the LPS treatment. Thus, membranes isolated from LPS-treated cells were enriched in ERp49 relative to those isolated from control cells.     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts.

Because knockout of the vimentin gene in mice did not produce an immediately obvious, overt, or lethal specific phenotype, the conjecture was made that the mutation affects some subtle cellular functions whose loss manifests itself only when the mutant animals are exposed to stress. In order to substantiate this idea in a tractable in vitro system, primary embryo fibroblasts from wildtype (V(+/+)) and vimentin-knockout (V(-/-)) mice were compared with regard to their growth behavior under the pseudophysiologic conditions of conventional cell culture. Whereas in the course of serial transfer, the V(+/+) fibroblasts progressively reduced their growth potential, passed through a growth minimum around passage 12 (crisis), and, as immortalized cells, resumed faster growth, the V(-/-) fibroblasts also cut down their growth rate but much earlier, and they either did not immortalize or did so at an almost undetectable rate. Cells withdrawing from the cell cycle showed increased concentrations of reactive oxygen species and signs of oxidative damage: enlarged and flattened morphology, large nuclear volume, reinforced stress fiber system as a result of increased contents of actin and associated proteins, prominent extracellular matrix, and perinuclear masses of pathological forms of mitochondria with low membrane potential. The differences in the cell cycle behavior of the V(+/+) and V(-/-) cells in conjunction with the morphologic changes observed in mitotically arrested cells suggests a protective function of vimentin against oxidative cell damage. Because vimentin exhibits affinity for and forms crosslinkage products with recombinogenic nuclear as well as mitochondrial DNA in intact cells, it is credible to postulate that vimentin plays a role in the recombinogenic repair of oxidative damage inflicted on the nuclear and mitochondrial genome throughout the cells' replicative lifespan. Recombinational events mediated by vimentin also appear to take place when the cells pass through the genetically unstable state of crisis to attain immortality. The residual immortalization potential of V(-/-) fibroblasts might be attributable to their capacity to synthesize, in place of vimentin, the tetrameric form of a lacZ fusion protein carrying, in addition to a nuclear localization signal, the N-terminal 59 amino acids of vimentin and thus its DNA-binding site. On the basis of these results and considerations, a major biologic role of vimentin may be to protect animals during development and postnatal life against genetic damage and, because of its contribution to the plasticity of the genome, to allow them to respond to environmental challenges.     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.     

140 000 Dalton surface glycoprotein : A plasma membrane component of the detergent-resistant cytoskeletal preparations of cultured human fibroblasts

A 140 000 D glycoprotein (140 kD gp), labelled radioactively with surface-specific techniques, remained as the major cell surface glycoprotein in the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. The 140 kD gp was present also in trypsinized cells and was not affected by treatment of the cells either with collagenase, chymotrypsin or thrombin. In density gradient fractionation of whole cells the 140 kD gp was recovered in the plasma membrane fraction together with small amounts of cytoskeletal components. In fractionation of cytoskeletal preparations, on the other hand, the 140 kD gp could not be dissociated from cytoskeletal proteins and together with vimentin it formed the major component of the oligomeric polypeptide complex generated by treating the surface-labelled cytoskeletal preparations with bifunctional cross-Linking reagent, dithiobis succinimidyl propionate (DTPS). Moreover, the 140 kD gp seemed to copurify with vimentin upon reconstitution of intermediate filaments from urea-solubilized cytoskeletal preparations. On the other hand, low ionic-induced degradation of vimentin led to a decrease in the amount of the detergent-resistant 140 kD gp on the cell surface. In electron microscopy, a close apposition between bilayer-like plasma membrane remnants of the adherent cytoskeletons and cytoskeletal elements could be seen. The results indicate that the 140 kD gp is a plasma membrane glycoprotein which closely interacts with the detergent-resistant cytoskeleton of cultured human fibroblast. Possible mechanisms of the association are discussed.     

Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus.

Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane.     

Proteins exposed at the adult schistosome surface revealed by biotinylation

The human blood-dwelling parasite Schistosoma mansoni can survive in the hostile host environment for decades and must therefore display effective strategies to evade the host immune responses. The surface of the adult worm is covered by a living syncytial layer, the tegument, bounded by a complex multilaminate surface. This comprises a normal plasma membrane overlain by a secreted bilayer, the membranocalyx. Recent proteomic studies have identified constituents of the tegument, but their relative locations remain to be established. We labeled the most exposed surface proteins using two impermeant biotinylation reagents that differed only in length. We anticipated that the two reagents would display distinct powers of penetration, thereby producing a differential labeling pattern. The labeled proteins were recovered by streptavidin affinity and identified by tandem mass spectrometry. A total of 28 proteins was identified, 13 labeled by a long form reagent and the same 13 plus a further 15 labeled by a short form reagent. The parasite proteins included membrane enzymes, transporters, and structural proteins. The short form reagent additionally labeled some cytosolic and cytoskeletal proteins, the latter being constituents of the intracellular spines. Only a single secreted protein was labeled, implying a location between the plasma membrane and the membranocalyx or as part of the latter. Four host proteins, three immunoglobulin heavy chains and C3c/C3dg, a fragment of complement C3, were labeled by both reagents indicating their exposed situation. The presence of the degraded complement C3 implicates inhibition of the classical pathway as a major element of the immune evasion strategy, whereas the recovery of only one truly secreted protein points to the membranocalyx acting primarily as an inert protective barrier between the immune system and the tegument plasma membrane. Collectively the labeled parasite proteins merit investigation as potential vaccine candidates.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.     

Direct cell injury associated with eutectic crystallization during freezing

Freezing induced direct cell injury has been explained by a two-factor hypothesis-intracellular ice formation (IIF) at rapid cooling rates, and solution effects at slow cooling rates. Even though IIF is generally believed to be a major injury mechanism at rapid cooling rates, injury by solution effects is not fully understood and several injury mechanisms have been suggested. Solution effects have generally been considered the result of the elevated electrolyte concentration within the intracellular and extracellular space during freezing. In addition to the injury by this elevated electrolyte concentration, freezing injury associated with eutectic crystallization was investigated. To examine the injury associated with eutectic crystallization, two different experiments were designed and performed. In the first experiment, two groups of AT-1 rat prostate tumor cell suspensions were frozen and thawed on a cryomicroscope in the same way except that eutectic crystallization was initiated in only one group. During the second experiment, AT-1 cells were suspended in several different media, which have different eutectic crystallization temperatures, and exposed to a single cooling-warming cycle with varying end temperature of the protocol on a directional solidification stage. After both experiments, post-thaw viability was evaluated and compared. The post-thaw viability drops significantly upon the occurrence of the eutectic crystallization regardless of suspending media, which suggests direct cell injury associated with eutectic crystallization. Based on these observations, two possible injury mechanisms are anticipated: (i) mechanical damage to the cell membrane due to eutectic crystallization, and (ii) intracellular eutectic formation (IEF). The proposed mechanisms provide a more comprehensive physical explanation of freezing induced cell injury and extend the understanding on solution effects.