Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography.

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate-fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.

Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C.

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.

A robotics-based automated assay for inorganic and organic phosphates.

Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.

Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C.

The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-delta1 in a continuous assay.

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface.

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.

Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C.

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GP) anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysaPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca(2+)-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein.

Photoelectron imaging of cells: photoconductivity extends the range of applicability.

Photoelectron imaging is a sensitive surface technique in which photons are used to excite electron emission. This novel method has been applied successfully in studies of relatively flat cultured cells, viruses, and protein-DNA complexes. However, rounded-up cell types such as tumor cells frequently are more difficult to image. By comparing photoelectron images of uncoated and metal-coated MCF-7 human breast carcinoma cells, it is shown that the problem is specimen charging rather than a fundamental limitation of the electron imaging process. This is confirmed by emission current measurements on uncoated monolayers of MCF-7 carcinoma cells and flatter, normal Wi-38 fibroblasts. We report here that sample charging in photoelectron microscopy can be eliminated in most specimens by simultaneous use of two light sources--the standard UV excitation source (e.g., 254 nm) and a longer wavelength light source (e.g., 325 nm). The reduction in sample charging results largely from enhanced photoconduction in the bulk sample and greatly extends the range of cells that can be examined by photoelectron imaging. The contributions of photoconductivity, the electric field of the imaging system, and the short escape depths of the photoelectrons combine to make photoelectron imaging a uniquely sensitive technique for the study of biological surfaces.

An extracellular inositol phospholipid-specific phospholipase C is released by cultured Swiss 3T3 cells.

A phosphatidylinositol-cleaving phospholipase C (PI-PLC) activity is released into the extracellular environment by intact Swiss 3T3 cell cultures. The activity is found in both serum-containing and serum-free defined culture medium. The cells remain attached and intact by Trypan Blue exclusion and lactate dehydrogenase assays. The activity is specific for phosphoinositides as no cleavage of phosphatidylcholine is observed. The activity is a phospholipase C rather than D since the water soluble products formed from cleavage of [3H]phosphatidylinositol were inositol phosphates and not inositol. Analysis of the inositol phosphate products showed a variation in composition with the pH of the assay, the ratio of noncyclic:cyclic forms being 60:40 at pH 7.5 and 40:60 at pH 5.5. This external phospholipase C resembles the well-characterized intracellular isozymes in that it is calcium-dependent and has a pH optimum between 5 and 6. From membrane filter assays the molecular weight of the native enzyme is estimated to be between 50 and 100 kDa.

Photoelectron imaging of viruses and DNA: evaluation of substrates by unidirectional low angle shadowing and photoemission current measurements.

Photoelectron imaging (photoelectron emission microscopy, PEM or PEEM) is a promising high resolution surface-sensitive technique for biophysical studies. At present, image quality is often limited by the underlying substrate. For photoelectron imaging, the substrate must be electrically conductive, low in electron emission, and relatively flat. A number of conductive substrate materials with relatively low electron emission were examined for surface roughness. Low angle, unidirectional shadowing of the specimens followed by photoelectron microscopy was found to be an effective way to test the quality of substrate surfaces. Optimal results were obtained by depositing approximately 0.1 nm of platinum-palladium (80:20) at an angle of 3 degrees. Among potential substrates for photoelectron imaging, silicon and evaporated chromium surfaces were found to be much smoother than evaporated magnesium fluoride, which initially appeared promising because of its very low electron emission. The best images were obtained with a chromium substrate coated with a thin layer of dextran derivatized with spermidine, which facilitated the spreading and adhesion of biomolecules to the surfaces. Making use of this substrate, improved photoelectron images are reported for tobacco mosaic virus particles and DNA-recA complexes.

Transition metal chelator TPEN counteracts phorbol ester-induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C.

Phorbol ester-induced reorganization of the actin cytoskeleton was investigated in C6 rat glioma cells. Observations by fluorescence microscopy and photoelectron microscopy indicated that pretreatment with the transition metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) for 1-2 h at 50 microM reduced the sensitivity of the actin cytoskeleton to disruption by the subsequent addition of 200 nM phorbol myristate acetate (PMA). The protective effect of TPEN was eliminated by adding back Zn2+ prior to PMA addition, implicating chelation of metal ions as the mechanism of action of TPEN. C6 cells exposed to PMA experience potent activation of protein kinase C (PKC) and substantial redistribution of the kinase from a soluble to a particulate cellular fraction (translocation). TPEN pretreatment did not block PKC translocation in PMA-exposed cells. By two-dimensional gel analysis, TPEN also did not reduce, but rather slightly increased, the PMA-stimulated phosphorylation of the acidic 80 kDa endogenous PKC substrate, as well as two other proteins at 18 kDa and 50 kDa. In contrast, TPEN significantly suppressed phosphorylation of a 20 kDa protein, both in cells treated with TPEN only and in TPEN-pretreated PMA-exposed cells. The results indicate that the ability of TPEN to protect against PKC-mediated actin cytoskeletal disruption is not due to either a block of PKC translocation or to general inhibition of PKC activity. Rather, the action of TPEN is more selective and probably involves chelation of Zn2+ at a critical Zn(2+)-dependent phosphorylation step downstream from the initial tumor promoter-induced effects on PKC.

Differential expression of phospholipase C specific for inositol phospholipids at the cell surface of rat glial cells and REF52 rat embryo fibroblasts.

Phosphatidylinositol(PI)-specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto-PLC) activity was calcium-dependent, did not result from secreted phospholipase C, and was not released from the cell surface by bacterial PI-specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L-glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto-PLC. The expression of ecto-PLC activity by REF52 fibroblasts was density-dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/10(6) cells), whereas in confluent cultures ecto-PLC activity increased to approximately 300 pmol/min/10(6) cells. In contrast to this behavior and that exhibited by previously reported ecto-PLC-positive cell types, the ecto-PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/10(6) cells) and by C6 glioma cells (approximately 100 pmol/min/10(6) cells) was independent of cell culture density up to confluence. The constitutive expression of ecto-PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.

Phorbol ester-induced actin cytoskeletal reorganization requires a heavy metal ion.

The cell-permeant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine(TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By using fluorescence and the higher resolution technique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 microM TPEN was found to dramatically reduce actin alterations resulting from subsequent phorbol ester treatment in PTK2 cells. Similar results were obtained with Swiss 3T3 cells using 50 microM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to depend on activation of protein kinase C (PKC). In contrast to the phorbol ester effect, the PKC-independent actin cytoskeletal disruption caused by staurosporine and cytochalasin B was unaffected by TPEN pretreatment. TPEN did not block phorbol ester-induced activation of PKC in Swiss 3T3 cells, as observed by the phosphorylation of the 80K PKC substrate protein (MARCKS protein). TPEN also did not inhibit partially purified PKC from Swiss 3T3 cells in an in vitro PKC-specific commercial assay. To establish that the effect of TPEN is the removal of metal ions and not some other nonspecific effect of TPEN, a series of transition metal ions was added at the end of the TPEN pretreatment. The results indicate that the transient but dramatic phorbol ester-induced reorganization of the actin cytoskeleton in cultured cells depends on an interaction of PKC with a heavy metal, probably zinc.

Biological applications of photoelectron imaging: a practical perspective.

Photoelectron imaging is finding a promising niche in the study of biological specimens. The features of photoelectron imaging that contribute to its uniqueness for this application are described. Image formation and the major contrast mechanisms of photoelectron microscopy, material contrast and topographical contrast are reviewed and illustrated with examples of photoelectron images of cultured cells and of DNA. General considerations in sample choice and preparation are also presented. Strategies for photoelectron labeling are discussed including the use of immunogold labeling, silver enhancement and cesium-based photocathodes.

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway.

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.

On the possibility of obtaining a physical map of genomes by photoelectron imaging.

Photoelectron imaging provides the possibility of a new method of mapping chromosomes. The basic concept is to cause DNA to emit electrons under the action of UV light. The criteria which must be met to map genomes by photoelectron imaging are set forth and discussed. Forming an image of the DNA by accelerating and focusing the electrons is a necessary but not sufficient condition for genome mapping. Equally important is to identify wavelengths of UV light which will cause selective emission from the base pairs, adenine-thymine and guanine-cytosine. The resulting image would then contain a modulation in the image brightness along the DNA duplex. By examining the photoelectron current from uniform films of homopolymers, a wavelength region is identified where marked differences in emission from base pairs is observed. At 160 nm, for example, the relative electron emission from a film of poly(dGdC) is approximately 5 times greater than for an equivalent film of poly(dAdT). Using the experimental data and known sequences, photoelectron gene maps are calculated for the bacteriophage lambda and for a short interspersed repetitive DNA sequence (an Alu repeat) of the human genome. The results suggest that a 5-nm physical map of chromosomes generated by photoelectron imaging would be informative and useful in mapping human and other large genomes.

Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells.

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 microM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 microM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 microM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.

Biological photocathodes.

Biological surfaces emit electrons when subjected to UV light. This emission is increased greatly after exposure to cesium vapor. Increases from 2 to 3 orders of magnitude are observed, depending on the biochemicals present. Heme and chlorophyll exhibit unusually high photoemission currents, which are increased further after cesiation. Photoemission from proteins and lipids is much less but also is increased by exposure to cesium. The formation of photocathodes with cesium greatly increases the practical magnifications attainable in photoelectron microscopy of organic and biological specimens. Photoelectron micrographs taken at magnifications greater than or equal to X 100,000 of chlorophyll-rich thylakoid membranes and of colloidal gold-labeled cytoskeleton preparations of cultured epithelial cells demonstrate the improvement in magnification. The selectivity and stability of the photocathodes suggest the possibility of detecting chromophore binding proteins in membranes and the design of photoelectron labels for tagging specific sites on biological surfaces.