Karen K. Hedberg

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.

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.

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.

Low‐energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system

Low‐energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow‐moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam‐separating system for LEEM and MEM.

Inhibition of the phosphatidylinositol-specific phospholipase C from Bacillus cereus by a monoclonal antibody binding to a region with sequence similarity to eukaryotic phospholipases.

Bacterial phosphatidylinositol-specific phospholipases C (PI-PLC) display similar substrate specificity as their eukaryotic counterparts involved in signal transduction of insulin and Ca2(+)-mobilizing hormones, and are used in the study of the novel glycosylphosphatidylinositol-protein anchors (GPI-anchors). For the investigation of structure-function aspects of the PI-PLC secreted from Bacillus cereus cells, a panel of murine monoclonal antibodies was generated and shown to be specific for the PI-PLC polypeptide in enzyme-linked immunosorbent assays and Western blots. Two of the monoclonals inhibited reactions catalyzed by the bacterial enzyme in vitro: hydrolysis of phosphatidylinositol and the release of bovine erythrocyte acetylcholinesterase from its GPI-anchor. At saturating concentrations of inhibitory antibody only a few percent of the enzyme activity remained. The epitope recognized by one of the inhibitory antibodies, A72-24, was mapped by proteolytic digestion, protein sequencing, and Western blotting of the generated fragments. The data indicate that at least part of the epitope resides within an 8 kDa-stretch of the bacterial PI-PLC (Gln-45 - Lys-122). Essentially the same segment of the bacterial polypeptide has previously been shown to display limited amino acid sequence similarity with several eukaryotic PI-specific phospholipases C (Kuppe, A., Evans, L.M., McMillen, D.A. and Griffith, O.H. (1989) J. Bacteriol. 171, 6077-6083). The results reported here suggest that the conserved peptide of these enzymes may contain functionally important residues.

Differential Expression of Phospholipase C Specific for Inositol Phospholipids at the Cell Surface of Rat Glial Cells and REF52 Rat Embryo Fibroblasts

Abstract: 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 phopholipase 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/106 cells), whereas in confluent cultures ecto‐PLC activity increased to approximately 300 pmol/min/106 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/106 cells) and by C6 glioma cells (approximately 100 pmol/min/106 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.

Immunophotoelectron microscopy of the cell surface and cytoskeleton.

A major goal of cell biology is an understanding of the structural and biochemical organization of the cell and how this organization relates to function. Electron microscopy provides one fruitful approach to this problem and has generated a wealth of structural information. A major advance took place in this field when immunolabeling technology (originally developed for immunofluorescence microscopy) was adapted to electron microscopy.’ The resulting technique of “immunoelectron” microscopy allows high-resolution identification of specific cellular components and their positions relative to other components. The familiar electron microscope techniques that make use of immunolabeling are transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Immunolabeling is also applied in an electron microscope technique that is a relative newcomer to cell biology, photoelectron microscopy (PEM).* PEM is a uniquely surface-sensitive electron microscopy that provides a different kind of view of the cell than TEM or SEM.334 The essential feature of PEM is that the image of the specimen results from UV light stimulated electron emission (photoemission) rather than from a transmitted, secondary, or backscattered electron signal resulting from exposure to a high-energy electron beam. Typical PEM biological samples are fixed, dehydrated whole mounts observed without staining, shadowing, or a conductive coating. Both the surfaces of cultured cells and cytoskeletal structures exposed after removal of the surface membrane are especially amenable to observation by this t e c h n i q ~ e . ~ In either case cellular organization is revealed primarily by topographical details, which provide a major source of contrast in PEM images! The application of immunolabeling in PEM makes use of a second source of contrast, photoemission contrast. Photoelectron labeling of biological structures depends on the use of markers that are more photoemissive than cellular components, rather than markers that are more electron-dense (as for TEM) or of a recognizable shape and size (as for SEM). The purpose of this paper is to illustrate the use of antibody-mediated photoelectron labeling (immunophotoelectron microscopy) using colloidal gold and silver-enhanced colloidal gold for the localization of specific cell surface and cytoskeletal proteins.

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.

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 (GPI)‐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 (lysoPI) 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 Ca2+‐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. J. Cell. Biochem. 65:550–564.