Circular-dichroism and fluorescence studies on melittin: effects of C-terminal modifications on tetramer formation and binding to phospholipid vesicles.

Studies were performed on a series of melittin analogues with selective alterations to the positively charged amino acid sequence at the C-terminus. Fluorescence studies were undertaken using the sole tryptophan residue in the analogues as an intrinsic fluorescence probe for indications of tetramer formation in free solution, and binding and insertion of the melittins into phospholipid bilayers. Studies were performed under conditions of low-salt buffer with increasing concentrations of phosphate added to promote self-association of the melittin monomers, and also in the presence of phospholipid vesicles. C.d. studies were also performed under conditions of increasing phosphate concentrations and in the presence of lipid vesicles to monitor the alpha-helical content of the melittins. It was found that selective replacement of the C-terminal basic amino acids by glutamine has different effects on self-association, alpha-helix formation and lipid binding of melittin.

Analysis of receptor clustering on cell surfaces by imaging fluorescent particles.

Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

Measurement of the rate of uptake and subcellular localization of porphyrins in cells using fluorescence digital imaging microscopy.

A fluorescence imaging system incorporating a cooled slow-scan charge-coupled device camera was used to study the rate of uptake and subcellular localization of prophyrins in living cells. Measurements were carried out on human dermal fibroblasts (D532) using two different prophyrins meso-tetra(4-N-methylpyridyl)porphine (TMPP) and meso-tetra(4-N-hexylpyridyl)porphine (THPP). It was observed that TMPP was rapidly taken up by cells and principally located in the nucleus. The THPP, on the other hand, internalized more slowly and exhibited a particulate distribution in the cytoplasm.

Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C.

A fluorescence imaging system, based on using a cooled slow-scan CCD camera, has been developed for tracking receptors on the surfaces of living cells. The technique is applicable to receptors for particles such as lipoproteins and viruses that can be labeled with a few tens of fluorophores. The positions of single particles in each image are determined to within 25 nm by fitting the fluorescence distribution to a two-dimensional Gaussian function. This procedure also provides an accurate measure of intensity, which is used as a tag for automated tracking of particles from frame to frame. The method is applied to an investigation of the mobility of receptors for LDL and influenza virus particles on human dermal fibroblasts at 4 degrees C. In contrast to previous studies by FRAP (fluorescence recovery after photo-bleaching), it is found that receptors have a low but measurable mobility at 4 degrees C. Analysis of individual particle tracks indicates that whilst some receptors undergo random diffusion, others undergo directed motion (flow) or diffusion restricted to a domain. A procedure is proposed for subdividing receptors according to their different types of motion and hence determining their motional parameters. The finding that receptors are not completely immobilised at 4 degrees C is significant for studies of receptor distributions performed at this temperature.

Digital fluorescence imaging of fusion of influenza virus with erythrocytes.

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow-scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20-22 degrees C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was less than 2 x 10(-13) cm2.s-1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.