James A. Dvorak

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.

Development and application of quantum dots for immunocytochemistry of human erythrocytes

Recent developments in quantum dot technology have resulted in the introduction of new fluorescence immunocytochemical probes. In contrast to organic fluorophores, which are not photostable, the high quantum yield and remarkable photostability of quantum dots solve major problems associated with immunocytochemical studies of erythrocytes. We report here the first application of quantum dots to immunocytochemical studies of human erythrocytes capable of being used in high‐magnification, three‐dimensional erythrocyte reconstruction techniques. The procedure consists of stabilizing human erythrocytes with a homofunctional imidoester cross‐linker to minimize fixative‐induced autofluorescence followed by reacting with a quantum dot – monoclonal antibody complex to label band 3 protein. Our new procedure clearly showed a non‐homogeneous, raft‐like distribution of band 3 protein in the erythrocyte membrane. We also demonstrate the applicability of our technique to studies of erythrocyte membrane modifications occurring during the invasion of a malaria parasite.

A controlled-environment culture system for high resolution light microscopy

Abstract A culture chamber has been developed within which cells or organisms can be maintained indefinitely in a controlled environment and studied by a variety of light microscopy techniques.

In Situ Compositional Analysis of Acidocalcisomes in Trypanosoma cruzi

We measured the elemental content of different compartments in Trypanosoma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron density contained (in units of mmol/kg dry weight ± S.E.) large amounts of phosphorus (1390 ± 13), magnesium (646 ± 19), calcium (171 ± 5), sodium (161 ± 18), and zinc (148 ± 6). No other compartment had appreciable calcium or zinc content. Iron (128 ± 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3- or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in the other elements investigated. This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes,i.e. acidic compartments rich in Ca2+. The acidocalcisomes were investigated by separation of epimastigote fractions on Percoll gradients in combination with Triton WR-1339 treatment. This detergent caused a rapid vacuolation; these vacuoles were shown by electron microscopy to be largely transparent, with a diffuse matrix. Percoll gradient fractionation demonstrated decreases in the density of various organelle markers in detergent-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as smaller decreases in the density of the other markers. Conventional electron microscopy of epimastigotes loaded with gold-labeled transferrin indicated that the endosomal system was separate from vacuoles that probably corresponded to the calcium-containing organelles detected by electron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites.

Trypanosoma cruzi: Interaction with vertebrate cells in vitro

Abstract Individual interactions between Trypanosoma cruzi trypomastigotes and vertebrate cells were observed in vitro as a continuous function of time under highly controlled environmental conditions. These observations form the basis for a description of the complete intracellular cycle of T. cruzi in susceptible vertebrate cells. Trypomastigotes actively penetrated the vertebrate cells. Multiple penetrations of a single cell were observed. A highly variable “lag period” occurred prior to the onset of parasite reproduction. This “lag period” was identical in two different host cell types. Amastigotes reproduced asynchronously, and the number of parasites produced in the host cell was directly proportional to the number of trypomastigotes penetrating the cell. The differentiation of amastigotes to trypomastigotes occurred by a direct “fusiform progression” pathway. The escape of trypomastigotes from the dead host cell occurred approximately 6.5 days post infection of the cell. Trypomastigotes were capable of infecting a vertebrate cell immediately after escaping from the cell in which they had completed their life cycle.

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 (β6: 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 μm) 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.

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.

Influence of monosaccharides on the infection of vertebrate cells by Trypanosoma cruzi and Toxoplasma gondii

The effect of 9 monosaccharides which constitute cell surface carbohydrates on the infection of bovine embryonic skin and muscle (BESM) cells by Trypanosoma cruzi trypomastigotes and Toxoplasma gondii tachyzoites was assayed. Most of the monosaccharides tested stimulated the infection of BESM cells by T. gondii; none of the monosaccharides were inhibitory. In contrast (at a concentration of 50 mM or greater) the monosaccharides inhibited non-specifically the infection of BESM cells by T. cruzi trypomastigotes whereas the other 8 monosaccharides were ineffective. The inhibition was due to an effect on the trypomastigotes and not on the vertebrate cells. It is proposed that there is a wheat-germ agglutinin-like lectin on the T. cruzi trypomastigote surface which recognizes and attaches to an N-acetylglucosamine-containing receptor on the vertebrate cell surface prior to infection. Infection of vertebrate cells by T. gondii tachyzoites appears to be mediated by other cell surface components. If monosaccharides are involved in infection by tachyzoites, they are ones not commonly found on animal cell surfaces. Alternatively, infection of vertebrate cells by T. cruzi and T. gondii is effected by different mechanisms.

Phase Imaging by Atomic Force Microscopy: Analysis of Living Homoiothermic Vertebrate Cells

Atomic force microscope-based phase imaging in air is capable of elucidating variations in material properties such as adhesion, friction, and viscoelasticity. However, the interpretation of phase images of specimens in a fluid environment requires clarification. In this report, we systematically analyzed atomic force microscope-derived phase images of mica, glass, and collagen under the same conditions as used for living cells at various tapping forces; the resulting data provide critical information for the interpretation of phase images of living cells. The peripheral regions of COS-1 cells consistently show a more negative phase shift than the glass substrate in phase images at set-point amplitude: free amplitude (Asp/A0) = 0.6-0.8. In addition, at all Asp/A0 values suitable for phase imaging, tapping frequency appears to be high enough to ensure that phase shifts are governed primarily by stiffness. Consequently, phase imaging is capable of high resolution studies of the cellular surface by detecting localized variations in stiffness. We demonstrate that phase imaging of a bifurcating fiber in COS-1 cell cytoplasm is readily capable of a lateral resolution of approximately 30 nm.

Trypanosoma cruzi: Interaction with mouse peritoneal macrophages

Abstract Interaction of the Ernestina strain of Trypanosoma cruzi with unstimulated BALE/ cAnN mouse peritoneal macrophages was observed continuously in a controlled-environment culture system. All motile forms of the parasite actively penetrated macrophages: trypomastigotes posterior end first, epimastigotes flagellar end first. Increased macrophage activity followed penetration. By 30 hr after penetration, trypomastigotes and “stationary phase” epimastigotes were destroyed. The macrophages died soon after the parasites were destroyed. By contrast, “exponential growth phase” epimastigotes could remain viable for up to 8 days within macrophages and subsequently died; the macrophages survived this interaction. Neither escape nor reproduction of intracellular parasites occurred in this system. During the period in which intracellular parasites were motile, the plasmalemma of the host cell was modified by penetration of the parasites flagellum. The possibility that these interactions are characteristic of specific strains of host and parasite only, could provide an explanation for the variability of T. cruzi infection seen in nature.