Surface Membrane Changes Research Articles

Preparation of Borrelia-Infected Mammalian Cells for Helium Ion Microscopy.

Preparation of mammalian cells for a Borrelia burgdorferi infection can be cumbersome especially if investigating possible cell entry processes. The initial steps of infection or entry into cells by a pathogen often involve attachment to the cell surface and plasma membrane changes. To topologically investigate with great resolution and detail these interactions of the pathogen and the mammalian cell, helium ion microscopy (HIM) can be employed. Here we describe a protocol used to define a specific multiplicity of infection (MOI) of Borrelia burgdorferi on a human chondrosarcoma cell line (SW1353) so that fine detail structures on the mammalian cell can be observed and quantified by HIM.

Editorial

Editorial

Leo Sachs. 14 October 1924—12 December 2013

Leo Sachs was a worldwide renowned scientist for his major contributions in several fields. In the mid 1950s he showed that amniocentesis could be used for prenatal diagnosis of sex and blood group antigens. He then focused on various aspects of normal development and cancer, including the control of normal haematopoiesis and leukaemia, carcinogenesis in vitro by polyoma and SV40 tumour viruses, chemical carcinogens and X-rays, chromosomes and the reversibility of cancer, surface membrane changes in malignant cells and suppression of malignancy by inducing differentiation. The cell culture system he established in the early 1960s for the clonal development of normal haematopoietic cells made it possible to analyse the molecular basis of normal haematopoiesis, and discover the proteins that regulate viability, proliferation and differentiation of different blood cell lineages and the changes that drive leukaemia. His studies established significant general concepts, including: the differential responsiveness of cancer cells to normal regulators of development; suppression of myeloid leukaemia by inducing differentiation, bypassing malignancy-driving genetic defects; identification of chromosomes that control tumour suppression; discovering apoptosis as a major mechanism by which WT-p53 suppresses malignancy; and the ability of haematopoietic cytokines to suppress apoptosis in both normal and leukaemic cells. Leo was fortunate to witness his pioneering discoveries and ideas move from the basic science stage to effective clinical applications, using amniocentesis for prenatal detection of genetic abnormalities, augmenting normal haematopoiesis in patients with various haematopoietic deficiencies and suppressing malignancy by inducing differentiation and apoptosis.

Physiological Changes of Surface Membrane in Lactobacillus with Prebiotics.

Synbiotics are always considered to be beneficial in healthy manipulation of gut environment; however, the purpose of this research was to investigate the dominance of synbiotic over the individual potential of probiotics and prebiotics. Four different types of prebiotics, fructo-oligosaccharides, raffinose, inulin, and cellobiose, were evaluated based on their varying degree of polymerization, combined each with 2 different Lactobacilli strains, including Lactobacillus paracasei 276 and Lactobacillus plantarum WCFS1. The effects of synbiotics combination on the surface structure were evaluated by analyzing auto-aggregation, membrane hydrophobicity, and adhesion to Caco-2 cells. Our results showed that both Lactobacilli exhibited significantly greater degree of attachment to Caco-2 cells (23.31% and 16.85%, respectively) when using cellobiose as a substrate than with other prebiotics (P < 0.05). Intestinal adhesion ability was in correlation with the percent of auto-aggregation, both Lactobacillus exhibited higher percent of auto-aggregation in cellobiose compared to other prebiotics. These behavioral changes in terms of attachment and auto-aggregation were further supported with the changes noticed from infrared spectra (FT-IR).

Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) During Infection of Erythrocytes in a Hamster Model

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.

Measurement of Changes in Membrane Surface Morphology Associated with Exocytosis Using Scanning Ion Conductance Microscopy

The extent that vesicles maintain a distinct identity and morphology after fusing with the plasma membrane is controversial. We used scanning ion conductance microscopy to image changes in the surface membrane of adrenal chromaffin cells after stimulation of exocytosis with a high K + solution. Within several minutes after stimulation, punctate depressions, 100–600 nm wide, were noted from 16% of the cells. The depressions were not randomly distributed, but appeared in clusters of two or more within a ∼1 μm 2 area and disappeared after several minutes. Increases in membrane surface area, consistent with the fusion and collapse of one or more vesicles into the surface membrane, were observed in 64% of the cells after high K + stimulation. Surface area increases did not occur if the high K + solution did not contain Ca 2+. We conclude that scanning ion conductance microscopy can be used to follow the time course of surface membrane changes resulting from exocytosis and endocytosis.

Incubation of Schistosoma mansoni lung-stage schistosomula in corn oil exposes their surface membrane antigenic specificities.

Several potential cues for increased surface antigenic expression of lung-stage schistosomula, such as lack of glucose and amino acids and extremes of pH or HCO3- concentration, failed to alter the negligible larval reactivity with control, infection, or irradiated cercariae-vaccine serum in indirect membrane immunofluorescence. In contrast, incubation of larvae in 90% corn oil for 6 hr led to surface membrane changes, which allowed specific and strong binding of antibody from antischistosome sera. The data together indicated that the lung-stage worms' confinement of antigenic molecules in lipid-rich sites of the outer membrane could be reversed in vitro after exposure to corn oil, in a concentration- and time-dependent manner.

Antineutrophil Cytoplasmic Antibodies Induce Reactive Oxygen-Dependent Dysregulation of Primed Neutrophil Apoptosis and Clearance by Macrophages

This study assessed whether anti-neutrophil cytoplasmic antibodies (ANCAs) interfere with the safe deletion of neutrophils by apoptosis and phagocytic clearance. Tumor necrosis factor (TNF)-primed neutrophils were incubated with normal IgG (N IgG) or ANCA IgG for up to 36 hours. Compared with N IgG, ANCAs accelerated constitutive apoptosis of TNF-alpha primed neutrophils, as assessed by morphology and confirmed by DNA laddering pattern on gel electrophoresis, and accelerated progression to secondary necrosis. The accelerated apoptosis induced by ANCA was dependent on reactive oxygen species generation, as primed neutrophils from patients with chronic granulomatous disease failed to show an effect of ANCAs on apoptosis. However, there was no change in the rate at which neutrophils exhibited annexin V binding, indicating that externalization of phosphatidylserine was not accelerated by ANCAs. Furthermore, when ANCA-treated primed neutrophils were interacted with human or murine peritoneal macrophages after 12 hours there was significantly less phagocytosis by human macrophages and no difference in phagocytosis by murine peritoneal-derived macrophages when compared with N IgG-treated controls. In conclusion, ANCAs accelerate apoptosis and secondary necrosis in TNF-primed neutrophils by a mechanism dependent on the generation of reactive oxygen species, with uncoupling of nuclear and surface membrane changes, resulting in a "reduced window of opportunity" for phagocytic recognition and engulfment before disintegration.

In vitro exposure of acute promyelocytic leukemia cells to arsenic trioxide (As2O3) induces the solitary expression of CD66c (NCA-50/90), a member of the CEA family.

Arsenic trioxide (As2O3) is a useful drug for the treatment of acute promyelocytic leukemia (APL), acting through a complex mechanism involving the induction of apoptosis. We investigated by flow cytometry whether in vitro treatment of APL leukemic cells with As2O3 determined specific surface membrane changes. Twelve APL bone marrow aspirates were analyzed following 7 days of in vitro treatment with As2O3 (0.25, 0.5 and 2.5 microM) with regard to the expression of a series of differentiation antigens. Twelve acute myeloid leukemia (AML) samples of non-APL morphotype were analyzed as controls. Exposure of APL as well as non-APL samples to any concentration of As2O3 did not affect the expression of beta2 integrins (CD11a and CD11b), CD45 isoforms (RA, RB and R0), CD44/H-CAM, CD33 and the CEA-related antigen family members CD66ade and CD66b, thus failing to disclose any maturating effect. Of interest, in all APL samples (but not in AML) every tested dose of As2O3 determined a dramatic upregulation of CD66c display; intermediate concentration (0.5 microM) of As2O3 increased the median percentage of CD66c+ cells from 5% in control cultures (25th-75th percentile 2-12%) to 80% in drug-exposed cultures (25th-75th percentile 58-90%) (P<0.001). The induction of solitary expression of CD66c is a new finding which demonstrates As2O3 capability of generating phenotypic changes absolutely restricted to APL cells Moreover, these results provide experimental basis for considering the involvement of the newly described CD66 signalling pathway in As2O3-driven programmed cell death.

Lateral mobility of proteins and lipids of cell surface membranes during aging: do the data support ‘The Membrane Hypothesis of Aging’?

Many previous studies regarding the change with age in surface membrane fluidity of different cell types, including hepatocytes, as determined by the fluorescence anisotropy method, are in conflict, demonstrating decreased, unchanged or even increased fluidity with age. In contrast, the results of our series of works using the fluorescence recovery after photobleaching (FRAP) technique, which measures protein lateral diffusion coefficients of hepatocyte surface membranes (Dp), have demonstrated that Dp generally declines in a linear fashion with age in hepatocytes of all animal strains and species examined. The major coworker (I. Zs.-Nagy) of these studies insists that our observations support his original hypothesis, ‘The Membrane Hypothesis of Aging’ (MHA), the primary assumption of which is that changes in cell surface membranes with age cause a general decline in intracellular enzyme activities. However, while it seems clear that cell surface membrane changes do occur with age, a number of past observations including those from the laboratory of this author, provide strong evidence that intracellular enzyme activities do not generally decline with age. This paper presents the data in detail, along with the author's view that the results do not support the main assumption of the MHA, but are more likely related to alterations in membrane functions with age.

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching.

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5-(octa-decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity.

Surface membrane remodeling following removal of vasopressin in toad urinary bladder

Vasopressin (ADH) increases transepithelial water flow in renal epithelia by a process that involves the insertion of water channels into the apical membrane. The objective of the present study was to examine membrane surface remodeling under conditions that promote the recovery of water channels. Hemibladders were set up as sacs with an imposed osmotic gradient. The control sacs received no hormone treatment, whereas the other sacs were stimulated with l00mU/ml ADH for 10 or 15 min to induce exocytosis and enhanced water flow. ADH was then washed from the tissues with fresh buffer rinses to abolish the hormone actions. These tissues were then allowed to recover for 15, 30 and 60 min. During this time water channels are recovered intracellularly by a process of endocytosis. This time period was called the retrieval period. At specified time intervals, tissues were fixed and processed for SEM or embedded in epon for ultrathin sectioning for TEM studies. Control tissues, regardless of the length of time, showed little or no sign of surface remodeling that was indicative of endocytosis during pre- or post-buffer washes, whereas the ADH- treated tissues showed a time-dependent remodeling of the apical membrane during activation and following removal of the hormone during the retrieval period. At the 10 min retrieval period, greater than 47% of the granular cells showed extensive surface remodeling. By 30 and 60 min post-hormone treatment during recovery, fewer than 23% of granular cells showed signs of surface membrane changes. During retrieval the apical membrane undergoes a transition with a loss of both microridges and microvilli prior to membrane restoration. These observations suggest that apical membrane remodeling is crucial for the restoration of membrane permeability following hormone activation and termination.

Cell surface membrane changes on differentiating neuroblastoma cells: [formula omitted] and [formula omitted]. Departments of Anatomy and Pathology, University of Rochester Medical Center, Rochester, N.Y. 14642

Cell surface membrane changes on differentiating neuroblastoma cells: [formula omitted] and [formula omitted]. Departments of Anatomy and Pathology, University of Rochester Medical Center, Rochester, N.Y. 14642

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

Anomalous cell surface structure of sickle cell anemia erythrocytes as demonstrated by cell surface labeling and endo-beta-galactosidase treatment.

Erythrocyte surface glycoproteins from patients with various types of sickle cell anemia have been analyzed and compared with those from normal individuals. By hemagglutination with various anti-carbohydrate antibodies, sickle cells showed profound increase of i antigens and moderate increase of GlcNAc beta 1 leads to 3Gal beta 1 leads to 3 Glc structure, whereas antigenicity toward globosidic structure was unchanged. In parallel to these findings, erythrocytes of sickle cell patients have additional sialylated lactosaminoglycan in Band 3. Thus, it can be concluded that erythrocytes of sickle cell patients are characterized by an altered cell surface structure which does not appear to be due to topographical changes of cell surface membrane. It is possible that the anemia or the "stress" hematopoiesis in these patients is responsible for these changes.

Surface-membrane biogenesis in rat intestinal epithelial cells at different stages of maturation.

The biosynthesis of membrane proteins and glycoproteins has been studied in rat intestinal crypt and villus cells by measuring the incorporation of L-[5,6-3H] fucose, D-[2-3H] mannose and L-[3,4,5-3H] leucine, given intraperitoneally, into Golgi, lateral-basal and luminal membranes. Incorporation of leucine and mannose was approximately equal in crypt and villus cells, whereas fucose incorporation was markedly higher (3-4 times) in the differentiated villus cells. As previously reported [Quaroni, Kirsch & Weiser (1979) Biochem J. 182. 203-212] most of the fucosylated glyco-proteins synthesized in the villus cells and initially present in the Golgi and lateral-basal membranes were found re-distributed, within 3-4h of label administration, in the luminal membrane. A similar process appeared to occur in the crypt cells, where, however, only few fucose-labelled glycoproteins were identified. In contrast, most of the leucine-labelled and many mannose-labelled membrane components found in the lateral-basal membrane of both crypt and villus cells did not seen to undergo a similar re-distribution process. The fucosylated glycoproteins of the intestinal epithelial cells represent, therefore, a special class of membrane components, most of which appear with differentiation, that are selectively localized in the luminal portion of the plasmalemma. In contrast with the marked differences in protein and glycoprotein patterns between the luminal membrane of villus and crypt cells, only minor differences were found between their lateral-basal membrane components: their protein patterns on sodium dodecyl sulphate/polyacrylamide slab gels, and the patterns of fucose-, mannose- and leucine-labelled components (analysed 3-4h after label administration) were very similar. Although the minor differences detected may be of importance, it appears that most of the surface-membrane changes accompanying cell differentiation in the intestinal epithelial cells are localized in the luminal portion of their surface membrane.

Interactions of gonadotropins with corpus luteum membranes: X. Biochemical and morphological changes in surface-membranes during PMSG-hCG induced luteinization of the rat ovary

Interactions of gonadotropins with corpus luteum membranes: X. Biochemical and morphological changes in surface-membranes during PMSG-hCG induced luteinization of the rat ovary

Ascaris oocytes: Ultrastructural and Immunocytochemical Changes during Passage through the Oviduct

Slender, bipolar, immature, Ascaris suum oocytes assume a smooth, spherical configuration as they traverse the oviduct. The present investigation was conducted to determine if this morphological alteration might be accompanied by changes in surface membranes. Immunocytochemical studies were conducted utilizing (1) fluorescein-labeled, sheep, anti-rabbit IgG, (2) Sternberger's unlabeled, antibody-enzyme technique, (3) ferritin-labeled, sheep, anti-rabbit IgG, and (4) an immunobead, labeled-antibody technique. These procedures clearly demonstrated that the surface of both immature and mature oocytes possessed different specific antigens. It is assumed that this acquisition of new antigens (or the loss of older ones) by the mature oocyte is somehow related to a conditioning phenomenon which prepares the oocyte for fertilization.

Asbestos body phagocytosis by human free alveolar macrophages

Phagocytosis of asbestos bodies by human free alveolar macrophages (FAMs) was documented employing light microscopy. This process was more carefully studied utilizing scanning electron microscopy (SEM) which demonstrated morphological and surface membrane changes in FAMs following phagocytosis of asbestos bodies. FAM viability was also evaluated following 24–72-h incubation of cells with asbestos bodies at a final concentration of 250 μg/ml. Slight, but significant, cytotoxicity was observed following the initial 24-h culture period ( P = 0.032, paired, 2-tailed t-test). No further cytotoxicity was observed, however, when cells were further incubated for 48-h and 72-h intervals ( P > 0.05 in all instances). These studies demonstrate asbestos bodies are readily phagocytized by cultured FAMs, and are only slightly cytotoxic to these human lung cells.

Changes of Surface Membrane and Chloroplast Ultrastructure of Barley, after Infection with Powdery mildew

Changes of Surface Membrane and Chloroplast Ultrastructure of Barley, after Infection with Powdery mildew