Altered membrane structure and surface potential in homozygous hemoglobin C erythrocytes.

BACKGROUND: Hemoglobin C differs from normal hemoglobin A by a glutamate-to-lysine substitution at position 6 of beta globin and is oxidatively unstable. Compared to homozygous AA erythrocytes, homozygous CC erythrocytes contain higher levels of membrane-associated hemichromes and more extensively clustered band 3 proteins. These findings suggest that CC erythrocytes have a different membrane matrix than AA erythrocytes. METHODOLOGY AND FINDINGS: We found that AA and CC erythrocytes differ in their membrane lipid composition, and that a subset of CC erythrocytes expresses increased levels of externalized phosphatidylserine. Detergent membrane analyses for raft marker proteins indicated that CC erythrocyte membranes are more resistant to detergent solubilization. These data suggest that membrane raft organization is modified in CC erythrocytes. In addition, the average zeta potential (a measure of surface electrochemical potential) of CC erythrocytes was approximately 2 mV lower than that of AA erythrocytes, indicating that substantial rearrangements occur in the membrane matrix of CC erythrocytes. We were able to recapitulate this low zeta potential phenotype in AA erythrocytes by treating them with NaNO(2) to oxidize hemoglobin A molecules and increase levels of membrane-associated hemichromes. CONCLUSION: Our data support the possibility that increased hemichrome deposition and altered lipid composition induce molecular rearrangements in CC erythrocyte membranes, resulting in a unique membrane structure.

The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte.

Using a combination of atomic force, scanning and transmission electron microscopy, we found that avian erythrocytes infected with the avian malaria parasite Plasmodium gallinaceum develop approximately 60 nm wide and approximately 430 nm long furrow-like structures on the surface. Furrows begin to appear during the early trophozoite stage of the parasite's development. They remain constant in size and density during the course of parasite maturation and are uniformly distributed in random orientations over the erythrocyte surface. In addition, the density of furrows is directly proportional to the number of parasites contained within the erythrocyte. These findings suggest that parasite-induced intraerythrocytic processes are involved in modifying the surface of host erythrocytes. These processes may be analogous to those of the human malaria parasite P. falciparum, which induces knob-like protrusions that mediate the pathogenic adherence of parasitized erythrocytes to microvessels. Although P. gallinaceum-infected erythrocytes do not seem to adhere to microvessels in the host chicken, the furrows might be involved in the pathogenesis of P. gallinaceum infections by some other mechanism involving host-pathogen interactions.

A Mycobacterium tuberculosis-derived lipid inhibits membrane fusion by modulating lipid membrane domains.

Tuberculosis is an infectious and potentially fatal disease caused by the acid-fast bacillus Mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. Lipoarabinomannan (LAM), an integral component of the MTB cell wall, is released when MTBs are taken into phagosomes and has been reported to be involved in the inhibition of phago-lysosomal (P-L) fusion. However, the physical chemistry of the effects of LAM on lipid membrane structure relative to P-L fusion has not been studied. We produced membranes in vitro composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol to simulate phagosomal lipid membranes and quantified the effects of the addition of LAM to these membranes, using fluorescence resonance energy transfer assays and atomic force microscopy. We found that LAM inhibits vesicle fusion and markedly alters lipid membrane domain morphology and sphingomyelin-chollesterol/dioleoylphosphatidylcholine ratios. These data demonstrate that LAM induces a dramatic reorganization of lipid membranes in vitro and clarifies the role of LAM in the inhibition of P-L fusion and the survival of the MTB within the macrophage.

A chemotactic response facilitates mosquito salivary gland infection by malaria sporozoites.

Sporozoite invasion of mosquito salivary glands is critical for malaria transmission to vertebrate hosts. After release into the mosquito hemocoel, the means by which malaria sporozoites locate the salivary glands is unknown. We developed a Matrigel-based in vitro system to observe and analyze the motility of GFP-expressing Plasmodium berghei sporozoites in the presence of salivary gland products of Anopheles stephensi mosquitoes using temperature-controlled, low-light-level video microscopy. Sporozoites moved toward unheated salivary gland homogenate (SGH) but not to SGH that had been heated at 56 degrees C for 30 min. We also investigated the origin of the attracted population. Attraction to SGH was restricted to hemolymph- and oocyst-derived sporozoites; salivary gland-derived sporozoites were not attracted to SGH. These data imply that sporozoites employ a chemotactic response to high molecular mass proteins or carbohydrate-binding proteins to locate salivary glands. This raises the possibility of utilizing anti-chemotactic factors for the development of mosquito transmission blocking agents.

Hemoglobin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes.

There is a well-established clinical association between hemoglobin genotype and innate protection against Plasmodium falciparum malaria. In contrast to normal hemoglobin A, mutant hemoglobin C is associated with substantial reductions in the risk of severe malaria in both heterozygous AC and homozygous CC individuals. Irrespective of hemoglobin genotype, parasites may induce knob-like projections on the erythrocyte surface. The knobs play a major role in the pathogenesis of severe malaria by serving as points of adherence for P. falciparum-infected erythrocytes to microvascular endothelia. To evaluate the influence of hemoglobin genotype on knob formation, we used a combination of atomic force and light microscopy for concomitant topographic and wide-field fluorescence imaging. Parasitized AA, AC, and CC erythrocytes showed a population of knobs with a mean width of approximately 70 nm. Parasitized AC and CC erythrocytes showed a second population of large knobs with a mean width of approximately 120 nm. Furthermore, spatial knob distribution analyses demonstrated that knobs on AC and CC erythrocytes were more aggregated than on AA erythrocytes. These data support a model in which large knobs and their aggregates are promoted by hemoglobin C, reducing the adherence of parasitized erythrocytes in the microvasculature and ameliorating the severity of a malaria infection.

Band 3 modifications in Plasmodium falciparum-infected AA and CC erythrocytes assayed by autocorrelation analysis using quantum dots.

The molecular stability of hemoglobin is critical for normal erythrocyte functions, including oxygen transport. Hemoglobin C (HbC) is a mutant hemoglobin that has increased oxidative susceptibility due to an amino acid substitution (beta6: Glu to Lys). The growth of Plasmodium falciparum is abnormal in homozygous CC erythrocytes in vitro, and CC individuals show innate protection against severe P. falciparum malaria. We investigated one possible mechanism of innate protection using a quantum dot technique to compare the distribution of host membrane band 3 molecules in genotypically normal (AA) to CC erythrocytes. The high photostability of quantum dots facilitated the construction of 3D cell images and the quantification of fluorescent signal intensity. Power spectra and 1D autocorrelation analyses showed band 3 clusters on the surface of infected AA and CC erythrocytes. These clusters became larger as the parasites matured and were more abundant in CC erythrocytes. Further, average cluster size (500 nm) in uninfected (native) CC erythrocytes was comparable with that of parasitized AA erythrocytes but was significantly larger (1 microm) in parasitized CC erythrocytes. Increased band 3 clustering may enhance recognition sites for autoantibodies, which could contribute to the protective effect of hemoglobin C against malaria.

Heterogeneous molecular distribution in supported multicomponent lipid bilayers.

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.

Atomic force microscopy of nanometric liposome adsorption and nanoscopic membrane domain formation.

Scanning probe microscopy studies of membrane fusion and nanoscopic structures were performed using hydrated single lipids and lipid mixtures. Extruded vesicles of DMPC and mixtures at various concentrations of DLPC, DPPC and cholesterol were deposited on freshly cleaved mica and studied in a fluid environment by AFM. The nanostructures formed by these extruded liposomes ranged from isolated unilamellar vesicles to flat sheet membranes and were marked influenced by thermodynamic phase behavior. For DMPC membrane, intact bilayers exhibited a phase transition process in agreement with large bilayer patches. In the DLPC, DPPC and cholesterol mixtures, nanoscopic domain diameters ranged from approximately 25 to 48nm with height differences of approximately 1.4nm; all values were lipid composition-dependent. Our data support and extend previous studies of microscopic domains and phase boundaries of the same mixtures in giant unilamellar vesicles determined by confocal light microscopy. Our approach for preparing and utilizing supported membrane structures is potentially relevant to studies of native cell membranes.

Induction of permeability changes and death of vertebrate cells is modulated by the virulence of Entamoeba spp. isolates.

Although Entamoeba histolytica is capable of inducing an apoptotic response in vertebrate cells in vitro (Cell. Microbiol. 2 (2000) 617), it is not known whether vertebrate cell death requires direct amoeba-vertebrate cell contact or simply the presence of amoebae in the area of the vertebrate cells. In addition, Entamoeba spp. vary in their virulence and pathogenicity. The potential effects of these critical parameters also have not been elucidated. We tested the virulent HM-1:IMSS isolate and the non-virulent Rahman isolate of E. histolytica, and the non-virulent E. dispar CYNO16:TPC isolate against two vertebrate cell lines, HeLa and Chinese hamster ovary cells in vitro using ethidium homodimer as a fluorescent indicator of changes in vertebrate cell permeability. Fluorescence appeared in vertebrate cell nuclei within approximately 2-3 min of contact between HM-1 amoebae and vertebrate cells independent of vertebrate cell type. However, vertebrate cells in the immediate vicinity of but not contacted by HM-1 amoebae were not affected. In contrast, although both E. histolytica Rahman and E. dispar CYNO16 amoebae moved freely among and contacted vertebrate cells, the nuclei of the vertebrate cells never fluoresced implying that the cells remained alive and impermeant to the ethidium homodimer. This is the first demonstration that direct contact between virulent amoebae and vertebrate cells is required to kill vertebrate cells and that the process is restricted to virulent Entamoeba isolates. An understanding at the molecular level of the processes involved could help to reduce the pathology associated with this parasite.

Nanoscopic lipid domain dynamics revealed by atomic force microscopy.

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.

The application of atomic force microscopy to the study of living vertebrate cells in culture.

Atomic force microscopy (AFM), a relatively new variant of scanning probe microscopy developed for the material sciences, is becoming an increasingly important tool in other disciplines. In this review I describe in nontechnical terms some of the basic aspects of using AFM to study living vertebrate cells. Although AFM has some unusual attributes such as an ability to be used with living cells, AFM also has attributes that make its use in cell biology a real challenge. This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.

Lipid membrane phase behaviour elucidated in real time by controlled environment atomic force microscopy.

Lipids are integral components of all biological membranes. Understanding the physical and chemical properties of these lipids is critical to our understanding of membrane functions. We developed a new atomic force microscope (AFM) approach to visualize in real time the temperature-induced lipid phase transition and domain separation processes in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes and estimate the thermodynamics of the phase transition process. The gel and liquid crystalline phases of DMPC coexisted over a broad temperature range (approximately 10 degrees C). Equal partitioning into two phases occurred at a transition temperature (Tm) of 28.5 degrees C. We developed a mathematical model to analyse AFM-derived DMPC membrane height changes as multi-peak Gaussian distributions. This approach allowed us to estimate the DMPC domain size, N, as 18-75 molecules per leaflet corresponding to a -4.2 nm diameter circular nanodomain. Lipid nanodomains may organize into microdomains or rafts which, in concert with proteins and other lipid components, play an important dynamic role in many biomedically important processes.

Detergent-resistant erythrocyte membrane rafts are modified by a Plasmodium falciparum infection.

Detergent resistant membranes (DRMs) have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. Flotillins-1 and -2 have recently been shown to be large components of erythrocyte DRMs. In this study, we show that a Plasmodium falciparum infection disrupts the association of flotillins with erythrocyte DRMs. Flotillins are probably released from erythrocyte DRMs through the reduction of cholesterol and sphingomyelin levels during the course of a P. falciparum-infection. Although it is well known that a P. falciparum infection can modify the host erythrocyte membrane, this is the first report that P. falciparum can alter the DRM components of erythrocyte membranes.

Estudios sobre la infección de ratones endogámicos con clones de Trypanosoma cruzi / Infection of inbred mice with Trypanosoma cruzi clones

El proyecto de investigación consistió en infectar cepas de ratones endogámicos con clones de Trypanosoma cruzi derivados del aislamiento de parásitos individuales y estudiar el curso de la infección. El proyecto aún no se ha terminado. Las etapas de la investigación consistieron en la determinación de: 1) el espectro de virulencia de los clones de T. cruzi; 2) la susceptibilidad relacionada con la cepa de los ratones a la infección del número de parásitos inoculados sobre el resultado de la infección con clones de T. cruzi; 4) la influencia de la vía de inoculación sobre el resultadso de la infección; 5) la influencia de la manipulación del parásito sobre el resultado de la infección; 6) la influencia de la infección por clon de T. cruzi sobre el resultado de la infección causada por un segundo clon, y 7) análisis cronológicos multiparamétrico del curso de la infección en un grupo grande de ratones, para el desarrollo de un modelo para la enfermedad de Chagas crónica

Estudios sobre la infeccion de ratones endogamicos con clones de Trypanosoma cruzi / Infection of inbred mice with Trypanosoma cruzi clones

The project, which is ongoing, consists of infecting strains of inbred mice with stocks of single-cell-isolated clones of Trypanosoma cruzi and studying the course and outcome of these infections. Research procedures included the determination of the following aspects: 1) virulence spectrum of T. cruzi clones; 2) mouse strain-dependent susceptibility to infection with T. cruzi clones; 3) effect of the number of parasites inoculated on the outcome of infection with T. cruzi clones; 4) influence of the route of inoculation on the outcome of infection; 5) influence of the handling of the parasite on the outcome of infection; 6) influence of the infection by one T. cruzi clone upon the outcome of infection by a second clone, and 7) a multiparametric, time series analysis of the course of infection in a large group of mice for the development of a chronic Chagas' disease model