Increased rhodamine 123 uptake by carcinoma cells.

The total cellular content of the fluorescent mitochondrial-specific dye rhodamine 123 (Rh-123) was quantified by butanol extraction as a function of time of exposure and dose for a variety of cell lines. These results were compared with observations made by fluorescence microscopy on dye localization and mitochondrial morphology. There appeared to be two categories of cell types based on Rh-123 uptake: those which progressively accumulate the dye, such as Ehrlich ascites tumor cells, carcinoma-derived lines MCF-7, PaCa-2, EJ, HeLa, and normal fibroblast line CCL 64; and those which appear to equilibrate with the extracellular dye within 1 h of incubation in Rh-123 (1 microgram/ml) with a minimal level of uptake, such as the normal epithelial-derived lines CV-1 and MDCK and the transformed fibroblast line 64F3. Within the first category, the absolute value of uptake per cell correlated with the concentration of Rh-123 in the medium and with the period of exposure to the dye up to a point of apparent cellular saturation. The length of time required for apparent saturation depended on the cell type. In the second category equilibration was very early, and the total uptake was a function of the extracellular concentration of Rh-123. This probably does not represent a saturation level of dye content in the non-accumulating, low uptake cell lines. Fluorescence microscopy revealed that Rh-123 localization was initially mitochondrial-specific for all of the cell lines examined. Over time, alterations in mitochondrial morphology and cytoplasmic fluorescence were observed in the high uptake cell lines but not in the minimal uptake cell lines. Incubation of the high uptake HeLa cell line with the mitochondrial membrane potential inhibitor p-trifluoromethoxyphenylhydrazone substantially decreased Rh-123 uptake. These observations may indicate a transformation-related characteristic of carcinoma cell mitochondria. It may be possible to exploit the mechanism responsible for the progressive accumulation of Rh-123 by carcinoma-derived cell types for chemotherapeutic approaches to certain types of carcinomas.

Photoelectron imaging of cytoskeletal elements.

The established electron microscope techniques have richly contributed to the current understanding of cytoskeletal structure. The purpose of this paper is to demonstrate the kinds of images of cytoskeletal structures obtained by photoelectron microscopy (PEM or photoelectron imaging), which has only recently been applied utilizes the fact that specimens exposed to UV light emit electrons (the photoelectric effect). These electrons are accelerated and focused to provide a detailed image of the exposed biological surface. There are several interdependent factors involved in imaging the cytoskeletons of cells grown in culture. Since PEM is a surface technique a major consideration is exposure of the structure of interest. A second consideration is the preservation of the three-dimensional integrity of the structures during the specimen preparation, and a third is preservation of antigenicity so that specific structures can be directly identified by antibody labeling methods. The photoelectron images of Triton-extracted cells reveal a detailed filamentous network of cytoskeletal elements. Prefixation of cells with a crosslinking agent (DTSP) prior to Triton extraction has little or no effect on the final image, whereas brief prefixation with 0.025% glutaraldehyde preserves more of the surface lamina and cytoplasmic organelles such as mitochondria. A comparison of immunofluorescence and photoelectron micrographs of the same cells shows a large correspondence in the fibers observed, and demonstrates the higher resolution of PEM. Direct identification of specific cytoskeletal elements in the photoelectron micrographs is illustrated using microtubule distributions in CV-1 cells by antibody decoration or, more generally, by the use of photoemissive gold markers on antibodies directed against microtubules.

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

The electron optical analog of immunofluorescence microscopy combines three developments: (i) photo-electron microscopy to produce a high-resolution image of exposed components of the cell, (ii) site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, provides a way to extend the useful immunofluorescence microscopy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were prepared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immunofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing enhanced photoemission from labeled cellular components so that they stand out against the darker background of the remaining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemission of the colloidal gold markers is sufficiently high that the microtubules are easily recognized without reference to the immunofluorescence micrographs, indicating that this approach can be used, in combination with antibodies, to correlate structure and function in cell biological studies.

The potential role of photoelectron microscopy in the analysis of biological surfaces.

The photoelectric effect provides the basis for an imaging technique useful for the study of biological surfaces. The photoelectron microscope (PEM) employs a UV lamp to photoeject electrons from the specimen surface. The electrons are then accelerated and imaged using electron optics. Photoelectron micrographs often resemble scanning electron micrographs, but the origin of contrast is different and these two techniques are complementary. Scanning Electron Microscopy (SEM) is unsurpassed in applications where specimens have pronounced relief or where elemental analysis is required. The advantages of PEM are a new origin of contrast, high sensitivity to fine topographical detail, short depth of information, and low specimen conductivity requirements. Photoelectron images of model systems, cell surfaces and cytoskeletal elements have been obtained.

A monoclonal antibody recognizing a keratin filament protein in a subset of transitional and glandular epithelia.

Monoclonal antibodies were raised against the detergent-insoluble cytoskeletal fraction of the human bladder carcinoma cell line, EJ. By immunofluorescence, one of these antibodies, H10-1, localized to keratin filaments in EJ and three out of five other transitional-cell carcinoma lines. Primary normal-human urothelial cells in culture were not recognized by H10-1. Strong staining of keratin filaments was also seen in 11 human adenocarcinoma cell lines, but it was only seen in a subpopulation of cells in one out of four squamous-cell carcinoma lines and not at all in two normal diploid human epidermal keratinocyte strains. Immunofluorescence on frozen sections of mouse, human, and rabbit tissues showed that H10-1 recognized the upper layer of the transitional epithelium of mouse and rabbit bladder, and a subset of simple epithelia (mouse stomach glands and some colon mucosal glands, but not kidney tubules, small intestine, ovary germinal epithelium, or endometrium; some human colon mucosal glands and glandular breast epithelium, but not endometrium). The antigen which is recognized by this antibody was not detected in examples of stratified squamous epithelium (mouse skin, nonglandular stomach, or esophagus; human skin, esophagus, or rectum) or nonepithelial tissue. On frozen sections of human tumors, the H10-1 antibody recognized 9 out of 14 colon carcinomas and 4 out of 6 breast carcinomas but did not recognize two sarcomas or a melanoma. This antibody apparently does not recognize its antigen in the presence of sodium dodecyl sulfate (SDS) because brief exposure of methanol-fixed cells to 0.1% SDS reversibly inhibited the binding of the H10-1 monoclonal antibody to keratin filaments by immunofluorescence, even when the binding of a rabbit polyclonal antikeratin antibody was unaffected. Two-dimensional gel-electrophoretic analysis of keratin-enriched fractions which had been isolated from various cell lines disclosed a good correlation between the presence of a 47-kdalton, pI-5.35 polypeptide and positive immunofluorescence with this antibody. These observations suggest that a certain keratin or keratin-associated protein is specifically expressed in a subpopulation of transitional and simple epithelial cells, and cultured cells derived from these epithelia. The monoclonal antibody described here may therefore be useful for increasing the precision of histological classification of epithelia, as well as the verification of the origin of certain cultured cell lines.

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.

Unusual retention of rhodamine 123 by mitochondria in muscle and carcinoma cells.

Mitochondria in cardiac muscle cells and myoblast-fused myotubes display unusually long (3-5 days) retention times of rhodamine 123, a mitochondria-specific fluorescent probe, in living cells. Among 50 keratin-positive carcinoma or transformed epithelial cell lines tested, mitochondria with prolonged rhodamine 123 retention are detected in most of the transitional cell carcinoma, adenocarcinoma, and chemical carcinogen-transformed epithelial cell lines and in some squamous cell carcinoma lines but not in any oat cell carcinoma lines. The presence of mitochondria having unusual dye retention may be useful for diagnosis and exploitable for chemotherapy of certain human carcinomas.

Photoelectron microscopy of cell surface topography.

Photoelectron micrographs of gold-palladium coated mouse 3T3 cells and chick embryo fibroblasts are presented. Since the gold-palladium suppresses differences in work function, the cell morphology seen in these micrographs is due to relief contrast. The heights of comparable cells were measured from the parallax present in transmission electron micrograph stereo-pairs of cell surface replicas. The origin of relief contrast and the effect of cell surface relief on the working depth of field in photoelectron images of cells are discussed. The micrographs demonstrate that photoelectron microscopy is very sensitive to fine details of cell surface topography, and that the working depth of field is not a limiting factor in the imaging of well-spread tissue culture cells.

Contrast effects in photoelectron microscopy; UV dose-dependent quantum yields of biological surface components.

The relative brightness of photoelectron microscopy images as a function of exposure to UV light has been determined from model systems representative of biological cell surface components. Quantitative data for amino acid homopolymers, yields. The photoelectron quantum yields, increase substantially over the initial values. For example, the quantum yields fo poly-L-tyrosine at 200 nm is initially about 5 X 10(-8) electron/incident photon. The quantum yield increases with 254 nm irradiation, leveling off at about 5 X 10(-4) electrons/incident photon after a dose of 3 X 10(21) quanta cm-2. Pre-irradiation of poly-L-tyrosine in the presence of certain chemical agents, for example, the Lewis base diborane (B2H6), results in a substantial reduction of the dose-dependent increase in quantum yield. Exposure to the reducing agent stannane (SnH4) essentially eliminates the effect. These chemical treatments provide methods of controlling the UV dose-dependent effects in the photoelectron images.

Photoelectron microscopy of cell surfaces.

Photoelectron micrographs of fixed, unstained, uncoated chicken embryo fibroblasts and absolute photoelectron quantum yields in the 180-230 nm wavelength band of L-fucose, D-galactose, D-glucose, N-acetyl-D-glucosamine, and the sucrose polymer Ficoll are reported. The quantum yields of the saccharides are low compared to the reference dye, phthalocyanine, and fall in the same range as those previously measured for amino acids and membrane phospholipids. Photoelectron micrographs of the unstained and uncoated cells inhibit considerable surface detail. The photoelectron quantum yield data and the micrographs indicate that surface relief is the dominant source of contrast.

Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes.

A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.