Marc Horisberger

COLLOIDAL GOLD, A USEFUL MARKER FOR TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.

Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope

A rapid method has been developed to visualize cell surface receptors in the SEM. Thus mannan at the surface ofCandida utilis cells was localized by stabilized colloidal gold granules coated with either antimannan antibodies or Con A.

Location of mannan and chitin on thin sections of budding yeasts with gold markers.

Mannan was located on thin sections of Saccharomyces cerevisiae and Candida utilis with the homologous anti-mannan antibodies or with Concanavalin A, both labelled with gold granules. Fully synthesized mannan was found in the cell walls, on the plasmalemma and within the cytoplasm sometimes associated with vesicles and vacuoles. Chitin or its oligomers were located with wheat germ agglutinin in the bud scars but also in the cell wall and the cytoplasm near the plasmalemma. Both mannan and chitin or its oligomers were found in the forming septum and are synthesized within the cytoplasm. The gold method was also suitable for marking mannan and chitin simultaneously.

Labelling of colloidal gold with protein A

SummaryMarkers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wise acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat β-lactoglobulin (βL) tritiated in vivo. When this protein was modified chemically by iodination with 125I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of βL, a maximum of 13–14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that βL was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-βL antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 βL molecules per particle could be completely bound by immobilized anti-βL antibodies. Preliminary data indicated that the energetics of adsorption of βL onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.

Ultrastructural localization of bowman-birk inhibitor on thin sections of Glycine max (Soybean) cv. Maple Arrow by the gold method

SummaryThe soybean Bowman-Birk inhibitor (BBI), a polypeptide of MW 8,000, has a specificity directed against trypsin and chymotrypsin. BBI was localized at the ultrastructural level by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledon and embryonic axis, BBI was found in all protein bodies, the nucleus and, to a lesser extent, the cytoplasm. Contrary to the Kunitz trypsin inhibitor (Horisberger and Tacchini-Vonlanthen 1983), BBI was not present in the cell wall but was found in the intercellular space. Intensity of marking in cotyledons of four-day-old seedlings was similar with the exception of the intercellular space which was free of BBI. In two lines lacking the Kunitz inhibitor (P.I. 157440 and 196168), data indicated that marking intensity was similar to that of cv. Maple Arrow. In contrast, in varieties lacking the lectin (Norredo, T-102) marking was more intense than in cv. Maple Arrow.

Colloidal gold as a tool in molecular biology

Abstract Monodisperse colloidal gold of different sizes (⩾5 nm) can be labelled with macromolecules which keep their bio-activities. These particles have found many applications as specific markers in scanning and transmission electron microscopy to identify, localize, quantify and understand the dynamics of cell components.

Location of glycoproteins on milk fat globule membrane by scanning and transmission electron microscopy, using lectin-labelled gold granules.

Abstract Membrane glycoproteins of bovine and human milk fat globules (MFG) were located by scanning electron microscopy using lectin-labelled gold granules (50 nm diameter) as specific markers. Receptors for wheat germ agglutinin (WGA) and soybean lectin (SBA) were localized in clusters over the whole MFG surface. When MFG were treated with neuraminidase, the density of marking with SBA increased. Marking of MFG with Concanavalin A (ConA) was weak. No marking was obtained with lectins specific for l -fucose, d -galactose and α- d -galactose. When thin sections of MFG marked with WGA (18 nm diameter gold granules) were examined by transmission electron microscopy, the membrane was uniformly marked. Using markers of different sizes (5 and 18 nm diam.) prefixed milk fat globule membranes (MFGM) were simultaneously marked with WGA and SBA. The lectin receptors appeared to belong to different glycoproteins which were clustered. Thin sections of this material showed that the receptors were located on one side of the membrane. No difference was observed between bovine MFG and human MFG from donors having blood group O and A. All results indicated that MFGM is a true biological membrane.

Localization of α-galactomannan and of wheat germ agglutinin receptors inSchizosaccharomyces pombe

The location of galactomannan on the surface ofSchizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized onS. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections ofS. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed thatS. pombe cell wall is devoid of chitin.

Ultrastructural localization of soybean agglutinin on thin sections of Glycine max (soybean) var. Altona by the gold method

SummarySoybean agglutinin (SBA) has been localized in Glycine max (soybean) var. Altona at the ultrastructural level by the gold method. SBA was detected by marking thin sections of different part of the seed with gold granules (12 nm in size) labelled with anti-SBA antiserum. Upon examination by transmission electron microscopy, the lectin was found uniformly distributed in most of the protein bodies of the cotyledon. SBA was also present in the embryo axis.

Mannan as a major component of the bud scars of Saccharomyces cerevisiae.

SummaryBud scars were prepared from the cell walls of Saccharomyces cerevisiae by means of an exo-β-d-(1→3)-glucanase. On acid hydrolysis, these structures were shown to consist of mannose (85%), glucose (4%) and glucosamine (2.7%). In addition the isolated bud scars were incubated either with mercurylabelled Concanavalin A or with Concanavalin A coated colloidal gold. They were then examined by electron microscopy and were found to be heavily marked, indicating the presence of mannan. It was thus shown that mannan is a true component of bud scars, which until now had been thought to consist solely of chitin and glucan.