Ultra scale-down studies of the effect of flow and impact conditions during E. coli cell processing.

The ability to recover cells from a fermentation broth in an intact form can be an important criterion for determining the overall performance of a recovery and purification sequence. Disruption of the cells can lead to undesired contamination of an extracellular product with intracellular components and vice versa loss of intracellular products may occur. In particular, the value of directed location of a product in the periplasmic space of say Escherichia coli (E. coli) would be diminished by such premature non-selective cell disruption. Several options exist for cell recovery/removal; namely centrifugation, in batch or continuous configuration, filtration or membrane operations, and in selected cases expanded beds. The choice of operation is dependant on many variables including the impact on the overall process sequence. In all cases, the cells are exposed to shear stresses of varying levels and times and additionally such environments exist in ancillary operations such as pumping, pipe flow, and control valves. In this study, a small-scale device has been designed to expose cells to controlled levels of shear, time and impact in a way that seeks to mimic those effects that may occur during full-scale processes. The extent of cell breakage was found to be proportional to shear stress. An additional level of breakage occurred due to the jet impacting on the collecting surface. Here it was possible to correlate the additional breakage with the impact velocity, which is a function of the distance that the jet travels before meeting the collection surface and the initial jet velocity.

Detection and spatial distribution of IL-2 receptors on mouse T-lymphocytes by immunogold-labeled ligands.

To identify the plasma membrane (PM) structures implicated in T-cell activation, we studied the distribution of interleukin-2 receptors (IL-2R) and the surface topography of lymphocytes by affinity labeling in electron microscopy (EM). In particular, we analyzed the distribution of the IL-2R alpha-chain on CTLL-2 cells (a murine cytotoxic T-cell lymphoma line). Some of our experiments were extended to the functionally and morphologically distinct cell line EL4 (a routine helper T-cell lymphoma line). As affinity ligands we used a rat monoclonal antibody (clone 7D4) reactive with the routine alpha-chain of IL-2R and recombinant mouse IL-2 (rIL-2). The distribution of IL-2R was visualized on the cell surface by ligands coupled to colloidal gold particles of different sizes. Unfixed cells were labeled with gold probes and attached to concanavalin A (ConA)-pretreated coverslips. Subsequently, the cells were prepared for EM. Examination of ultrathin sections and large surface replicas revealed a high degree of variability in cell morphology and in the density of the randomly distributed gold-labeled ligands among CTLL cells. According to their typical appearance, lymphocytes with strong receptor expression can be easily identified within the cell population. In contrast, the label on many mitogen-activated EL4 cells showed a cap-like polar distribution. The results suggest the existence of diverse distribution patterns of IL-2R on CTLL and EL4 cells. These differences are believed to reflect the different physiological roles played by T-cell subsets in the immune system.

Distribution and ultrastructure of plectin arrays in subclones of rat glioma C6 cells differing in intermediate filament protein (vimentin) expression.

Histochemical and biochemical studies suggest that the functions of the intermediate filament (IF) binding protein plectin comprise the physical linkage of IFs to each other and to other cytoskeletal elements, and their anchorage at membrane-attached junctional complexes. To further evaluate this hypothesis the expression, cellular distribution, and ultrastructure of plectin arrays were studied in rat glioma C6 cell subclones differing in IF protein (vimentin) expression. Here we show that plectin is expressed in a vimentin-negative C6 cell subclone (C6-D10) at levels similar to those of the vimentin-positive control subclone C6-D8. However, the amount of cytoskeleton-associated plectin found after extraction of cells with Triton X-100 or Triton X-100/high salt was significantly reduced in IF-negative compared to IF-positive cells. Using immunofluorescence microscopy, plectin structures were detected throughout the cytoplasm of IF-deficient cells. Unlike in IF-containing cells, where plectin colocalized largely with the vimentin network, in the IF-negative subclone the protein was mainly associated with polymeric actin structures. The release of plectin from IF-deficient cytoskeletons upon treatment with heavy meromyosin argued for specificity of the plectin microfilament interaction. Whole mount electron microscopy in conjunction with immunogold labeling of cytoskeletons revealed that in both IF-positive and IF-negative cells, plectin label specifically associated with thin (3-nm) filamentous structures that were clearly distinct from the major cytoskeletal filament systems. In IF-containing cells these filaments were found to link IFs to actin filaments and to connect vimentin filaments to each other. In IF-deficient cells, filamentous plectin structures were found to form dense cytoplasmic networks together with actin filaments and actin filament bundles. These data support the hypothesis that filamentous plectin arrays play an important role in the structural organization and mechanical integration of the cytoskeleton, in particular IFs and microfilaments.

High-pressure freezing of cell suspensions in cellulose capillary tubes.

A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro-organisms by high-pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 microns, into which the suspensions are drawn by capillary action. The tubes are processed by high-pressure freezing and freeze-substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated. A very high yield of adequately frozen specimens is obtained due to the constant, defined sample geometry. This approach can also be used to process suspensions by conventional chemical fixation, eliminating the need to embed pellets in low-melting-point agarose, for example, prior to chemical fixation. The preparation procedure is demonstrated with suspensions of nematodes, paramecia and bacteria.

Cytoskeleton architecture of C6 rat glioma cell subclones differing in intermediate filament protein expression.

Whole-mount electron microscopy was used in conjunction with immunogold labeling to characterize the cytoskeleton architecture of C6 rat glioma cell subclones. These subclones differ in intermediate filament (IF) protein composition and either contain vimentin (subclone C6D8) or do not express any of the known cytoplasmic IF proteins (subclone C6D10) (Röser et al., 1991). In C6D8 cells short thin (3 nm) connecting filaments frequently linked vimentin to actin filaments and, in addition, connected vimentin filaments to each other. Occasionally, direct contacts were noticed between actin and vimentin filaments. Thin connecting filaments were present at a significantly higher number in IF-deficient C6D10 cells, forming a dense cytoplasmic network in conjunction with actin filament bundles as the dominating structure. The data indicate that thin connecting filaments are present in C6 cells independent of the expression of cytoplasmic IF proteins. They suggest that structural linkages between vimentin and actin filaments mediated by thin connecting filaments could play a major role in determining the cytoskeleton architecture of these cells.

Subclones of C6 rat glioma cells differing in intermediate filament protein expression.

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Immunoelectron microscopy on the topographical distribution of the poliovirus receptor.

The topographical distribution of the poliovirus receptor on the cell surface was demonstrated by immunoelectron microscopy using monoclonal antibodies and immunogold markers. The receptor appeared in small clusters, which were randomly distributed over the cell surface and along cellular processes. The distribution pattern of the clusters corresponded to that of absorbed and immunogold-labelled poliovirus particles and suggests a multivalent organization of poliovirus binding sites. Freeze-fracturing and ultrathin sectioning did not reveal any specific ultrastructures within the plasma membrane at labelled receptor areas. Incubation of native cells with anti-receptor antibodies did not remove the receptor molecule from the cell surface nor did it induce ultrastructural alterations within the plasma membrane. The antibody-receptor complexes exhibited lateral mobility within the plasma membrane and were able to aggregate into large immune complexes after incubation with a second ligand.

Localization of nucleocapsid associated polypeptides in measles virus-infected cells by immunogold labelling after resin embedding.

The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.

Morphogenesis of measles virus on C6 rat glioma cells.

Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.

Demonstration of antigens at both sides of plasma membranes in one coincident electron microscopic image: a double-immunogold replica study of virus-infected cells.

We present here a procedure for obtaining high-resolution topographical information about the spatial distribution of antigens at both sides of isolated plasma membranes. HeLa cells grown on coverslips and infected with measles virus served as a model system. Virus glycoproteins appearing at the cell surface were demonstrated by tagging them with rabbit anti-measles antibodies and protein A-gold probes. Cells were stabilized with tannic acid, covered with a cationized coverslip, and then split in potassium-containing buffer. Membranes adherent to the cationized coverslip were fixed in formaldehyde-glutaraldehyde and reacted with mouse monoclonal antibodies against various structural proteins of measles virus. Antibody binding sites at the cytoplasmic surface were visualized either by the antibody bridge method, using normal mouse Ig coupled to gold colloid of different sizes, or by the peroxidase-antiperoxidase procedure. After osmication and critical point-drying, the cytoplasmic surfaces were replicated by platinum-carbon evaporation and examined by TEM without prior cleaning from biological material. This new method permits concomitant localization of antigens present at the inner and outer leaflets of the plasma membrane, and provides high-resolution information about the three-dimensional organization of the cytoplasmic surface.

Replica-immunogold technique applied to studies on measles virus morphogenesis.

The replica technique was applied to studies on the dynamic process of measles virus budding on infected HeLa cells. Virus structures were identified by labeling with anti-measles antibodies and protein A-gold. The combination of these two methods enabled us to characterize the sequence of virus budding at the plasma membrane, to localize virus structures on cytoskeletons of infected cells, and to study the influence of Ca2+ ions on virus structures at the plasma membrane. Studies on platinum carbon surface replicas suggest that the process of virus budding is similar to the genesis of cellular microvilli. Replicas prepared from cytoskeletons of infected cells reveal a close association of budding virus with actin filaments composing the outer parts of the networks. Replicas of apical plasma membranes isolated from infected cells show the attachment of viral nucleocapsids to the protoplasmic membrane face of infected cells. These nucleocapsids are not present on membranes prepared from cells treated with calcium and the ionophore A23187. In addition viral cell surface antigens become randomly distributed on these cells. The data suggest that measles virus morphogenesis at the plasma membrane of cultured cells is dependent on the function of the cytoskeleton and may be influenced by Ca2+ ions.

Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells.

Cytoskeletons were prepared from measles virus infected HeLa cells to investigate the involvement of cytoskeletal filaments in virus budding at the plasma membrane. The cytoskeletons retained nearly 80% of measles virus hemagglutinin, the major viral polypeptides, including P, NP, and M, and 2 to 12% of the total cell bound infectivity. As demonstrated with platinum- and carbon-shadowed cytoskeletons, all stages of budding, i.e., virus specific strands, stub-like protrusions, and completely rounded virus particles, are associated with actin filaments composing the outer part of the cytoskeletal network. As shown with ultrathin sections of flat embedded extracted cells, actin filaments identified with heavy meromyosin almost exclusively protrude into virus particles with their barbed ends and are in close association with viral nucleocapsids. The data support previous suggestions that actin is involved in virus budding and show that budding itself is possibly the result of a vectorial growth of actin filaments.

Preparation of apical plasma membranes from cells grown on coverslips. Electron microscopic investigations of the protoplasmic surface.

We introduce here a simple method which permits an efficient isolation of apical plasma membranes from tissue culture cells and the electron microscopic examination of their protoplasmic surfaces by use of the platinum/carbon replica technique. Different procedures were tested with regard to the efficiency of isolation and preservation of ultrastructure. Best results were obtained by prestabilization of cell surfaces with low concentrations of tannic acid prior to isolation. To demonstrate the possible applications and versatility of the method, studies were done on virus-infected cells in combination with immunocytochemical labeling. With this model system, we show that it is possible to correlate the structures seen on the cytoplasmic surface of the plasma membrane with the distribution of virus antigens at the cell surface labeled with immunogold markers prior to preparation.

Immunochemical study on the contribution of hypolipidaemic-induced cytochrome P-452 to the metabolism of lauric acid and arachidonic acid.

The influence of four hypolipidaemic drugs (clofibrate, WY-14,643, clobuzarit and bezafibrate) on hepatic cytochrome P-450 and fatty acid metabolism in male rat liver microsomes has been investigated. All of the hypolipidaemic drugs tested significantly induced the hydroxylation of lauric acid and, furthermore, this was accompanied by a concomitant 3-fold induction of a specific isoenzyme of cytochrome P-450 (termed cytochrome P-452) as determined by a single radial immunodiffusion technique. In addition, immunochemical quantitation of cytochrome P-452 in control, uninduced rat liver microsomes revealed that this particular isoenzyme constituted 22% of the total carbon monoxide-discernible cytochrome P-450 population. This has led us to the conclusion that cytochrome P-452 is a constitutive cytochrome P-450 isoenzyme and therefore that hypolipidaemic agents function as inducers of constitutive haemoprotein isoenzymes. Cytochrome P-452 plays a significant role in the hydroxylation of lauric acid as evidenced by inhibition of hydroxylase activity in the presence of an anti-P-452 IgG fraction. In addition, this antibody preferentially inhibits the 12-hydroxylation of lauric acid in rat liver microsomes by comparison to the 11-hydroxylase activity. Our studies have also shown that arachidonic acid serves as an excellent substrate for hypolipidaemic-induced cytochrome P-452, resulting in the formation of several metabolites that have been separated by reverse phase HPLC. Furthermore, a specific metabolite (or group of metabolites) of arachidonic acid is induced by clofibrate pretreatment and that the formation of this metabolite(s) is inhibited by an antibody to cytochrome P-452. By comparison, other metabolites of arachidonic acid remain refractory to induction by clofibrate and are not inhibited by the presence of anti-P-452 IgG. In addition, a reconstituted enzyme system containing highly purified cytochrome P-452 actively catalyses the above specific oxidation of arachidonic acid, a reaction that is significantly stimulated by the presence of cytochrome b5. Taken collectively, our data provide compelling evidence that hypolipidaemic agents induce a specific isoenzyme of hepatic microsomal P-450 that readily oxidizes fatty acids and that arachidonic acid may serve as an excellent endogenous substrate for this novel haemoprotein.

Inhibition of measles virus budding by phenothiazines.

HeLa cells infected with measles virus show an accumulation of virus-specific strands at the plasma membrane after addition of the anticalmodulin drugs trifluoperazine (TFP) and chlorpromazine (CPZ), whereas spherical virus particles are almost completely absent. At low drug concentrations (10-15 microM TFP; 30-40 microM CPZ) the inhibitory effect is dependent on the presence of extracellular calcium. The strands complete the budding process after removal of the drugs. Restoration of virus budding is not sensitive to cycloheximide and immunoprecipitation experiments give evidence that the viral protein synthesis is not qualitatively altered in the presence of TFP. The data indicate that both drugs arrest the budding process at an intermediate stage at the plasma membrane. The inability of the strands to comigrate with cytochalasin B-induced actin patches suggests that the inhibition of budding is probably the result of an impaired interaction of viral structures with the cytoskeleton.

Protein-A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces.

Due to their high atomic number contrast in transmission electron microscopy, gold particles are ideal markers in surface replicas of cultured cells. The suitability of protein-A-coated gold particles in replica immunocytochemistry for labelling surface antigens is demonstrated using measles virus-infected cells as a model system. Labelled areas can easily be distinguished from unlabelled areas, and even markers positioned in the evaporation shadow of large structures can be accurately identified, which is a prerequisite for an exact quantification and mapping of antigen. In addition, the ultrastructure of labelled areas can still be visualized because of the small size of the marker.