Ehud Skutelsky

Use of cationized ferritin as a label of negative charges on cell surfaces.

A polycationic derivative of ferritin for labeling of negative charges on cell surfaces was prepared by coupling horse spleen ferritin with N, N dimethyl-1,3 propanediamine (DMPA) via carbodiimide activation of the protein carboxyl groups. Various derivatives of cationized ferritin prepared in the pH range between 5 and 7 were characterized and tested for their labeling capacity. The DMPA-ferritin prepared at pH 6.5 was chosen as the optimal preparation for the present study. Labeling with cationized ferritin is performed at physiological conditions with or without prefixation with glutaraldehyde. It is effective on a variety of cells, and its geometry permits relatively easy counting on the membrane surface. The difference in surface charge labeling between young, old, and receptor-destroying enzyme (RDE)-treated rabbit red cells is reflected in the number of cationized ferritin particles attached per unit area of membrane in tangentially sectioned red cells. This is in agreement with the values obtained by electrophoresis.

The avidin-biotin complex in affinity cytochemistry.

Publisher Summary This chapter describes the avidin–biotin complex in affinity cytochemistry. The use of affinity methods for the localization, visualization, and subsequent evaluation of specific cellular components is termed “affinity cytochemistry.” In general, the technique is based upon the preparation of a mixed conjugate, comprising a biologically active molecule attached chemically to a potentially perceptible probe, whereby the resultant product retains both detectability and biological activity. A wide spectrum of biologically active molecules is coupled to the above probes, including antibodies. The use of the high-affinity avidin–biotin complex is shown to circumvent some of the problems relating to ferritin–protein conjugation. In addition, this method is employed to unify and facilitate certain aspects of affinity cytochemical techniques. There are numerous steps involved in this step, such as biotin is attached via an appropriate reactive derivative either directly to cell surface functional groups or to a biologically active molecule. The preparation of ferritin–avidin conjugates is described in the chapter.

Affinity cytochemistry: The localization of lectin and antibody receptors on erythrocytes via the avidin-biotin complex

Ferritin-conjugated proteins, such as lectins and antibodies, are widely used for localization of cell surface receptors in the electron microscope [ 1,2] . Procedures designed to effect direct covalent coupling of ferritin to the binding-protein are, for the most part, cumbersome and inefficient. The resultant complex is of high molecular weight often a multimer thus affecting both the physical and chemical binding characteristics and the biological activity of the conjugate. Biotin, derivatized to bacteriophages via the terminal carboxy moiety, has previously been shown to be available for subsequence interaction with avidin, resulting in phage inactivation [3] . A later investigation demonstrated that ferritin-avidin conjugates can be used for the specific labeling of biotin-tagged proteins and membrane preparations [4]. Recently, we have described an improved technique for the preparation of ferritin-avidin conjugates for use as an ultrastructural marker in the localization of surface receptor sites on biotin-derivatized whole cells [S] . The visualization of tRNA hybridized to DNA has also been reported [6].

On the mode of liposome-cell interactions. Biotin-conjugated lipids as ultrastructural probes

An efficient method for labeling and visualizing phospholipids at the ultrastructural level is described. Biotin was coupled to the amines of appropriate phospholipids via the N-hydroxysuccinimide ester. The biotinylated lipid could be specifically labeled by ferritin-avidin conjugates and detected by transmission electron microscopy. The lipid derivatives were analyzed and evaluated in terms of their resemblance to the original lipid. Although differing in some aspects from the parent lipid molecules, the biotinyl derivatives still retain the basic characteristics of lipids vis-a-vis their orientation and position in the membrane bilayer. The latter property renders the biotinylated lipid qualitatively suitable for tracing the fate of the lipid component(s) of liposomes during their interaction with biological membranes of various cell types. Using this system, we propose that the extent and pattern of the liposome-cell interaction depends, at least in part, on the cell type employed. This observation may be due to intrinsic variations in cell surface structure and properties relative to those of the liposome.

Surface charge properties of the luminal front of blood vessel, walls: An electron microscopical analysis

Abstract The negative charge density and distribution on cell membranes of vascular endothelium and subendothelial components of Guinea pig and rabbit blood vessels were evaluated using cationized ferritin (CF) and positively charged colloidal iron (CI). Isolated segments of vena cava and aorta as well as washed RBCs were fixed with aldehydes, washed and labeled with CF at pH 7.2 or with CI at pH 1.8. The particles were evenly distributed on the luminal front of the vascular endothelium. CF was seen on perpendicular sections about 20 A from the plasmalemma. No significant difference was detected between the charge density of vascular endothelium of Guinea pig and rabbit, despite a striking difference in the charge density of the respective RBCs. Treatment with neuraminidase resulted in 50% reduction of anionic groups on endothelial cells and 80% reduction RBCs. Subendothelial components including basement membrane, microfibrils and collagen fibers were densely labeled with CF and CI before and after treatment with neuraminidase. The results suggest that specific receptors rather than reduction in surface charge play a role in adhesion of thrombocytes to subendothelial components.

Localization of sialyl residues on cell surfaces by affinity cytochemistry

The use of the avidin—biotin complex for the specific ultrastructural visualization and evaluation of cell surface sialyl residues was investigated. Circulating red blood cells of various mammalian species and bone marrow cells of the rabbit were treated with sodium meta-periodate and biotin hydrazide in succession, with or without prior treatment of the cells with neuraminidase. The results were compared with the distribution of cell surface anionic sites, estimated by the rate of agglutination with poly- l -lysine and the binding capacity of positively charged colloidal iron and cationized ferritin. A uniform labeling of the erythrocyte surface was obtained with variations between different animal species. Neuraminidase treatment drastically reduced the sensitivity of cells to periodate-induced biotinylation. This method represents a distinct improvement over past approaches, since it can be applied to unfixed cells at physiological pH. An additional feature of this system enabled the topographic resolution of sialic acid residues from the outer dense line of the erythrocyte membrane. A value of 50–70 A was obtained for human erythrocytes. Developmental and species-dependent modulations in the proximity of sialic acid to the membrane were also observed. The frequency of attached ferritin—avidin conjugates to biotinylated erythroid cells in rabbit bone marrow was found to vary according to the degree of maturation in the erythroid line, corresponding to the previously described variations in colloidal iron binding.

PREPARATION OF FERRITIN-AVIDIN CONJUGATES BY REDUCTIVE ALKYLATION FOR USE IN ELECTRON MICROSCOPIC CYTOCHEMISTRY

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiffs bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.

Distribution of the T-antigen on erythroid cell surfaces. Studies with peanut agglutinin, an anti-T specific lectin

Abstract The interaction of peanut agglutinin, a lectin with a specificity similar to that of serum T-agglutinin, with human, guinea pig and rabbit young and old red blood cells and bone marrow erythroid cells, both before and after neuraminidase treatment, was investigated. Distribution of the peanut agglutinin receptors was determined by electron microscopical analysis with the ferritin conjugated lectin. In addition, kinetics of agglutination by the lectin were examined with the aid of the Fragiligraph. Rabbit red blood cells of all ages bound peanut agglutinin without neuraminidase treatment. Untreated human and guinea pig red blood cells, as well as immature erythroid cells of guinea pig bone marrow were not agglutinated by the lectin, nor was binding of the lectin to these cells observed by electron microscopy. Treatment with neuraminidase resulted in exposure of peanut agglutinin binding sites on erythroid cells of all stages of maturation and aging. Our findings show that peanut agglutinin receptors are absent from the surface of old circulating red blood cells and extruded erythroid nuclei, in spite of a remarkable in vivo loss of surface sialyl residues. The use of neuraminidase-treated red blood cells, as a model for the study of the clearance of aged red blood cells from circulation seems, therefore, not to be entirely justified.

Electron Microscopical Analysis of Surface Charge Labelling Density at Various Stages of the Erythroid Line

SummaryWith use of the positively charged, colloidal ferric oxide labelling technique for electron microscopy of sections of rabbit marrow, a reduction in labelling density on the surface of differentiating red cell precursors was demonstrated. The number of iron particles per unit length of membrane was counted. A progressive diminution in labelling density follows cell division, reaching a minimum in the orthochromatic erythroblast, from which the nucleus is expelled. A slight increase in charge density is noted in the reticulocyte, and further increase is observed with its maturation to the erythrocyte. The results indicate that biosynthesis of n-acetyl neuraminic acid stops at the earliest recognizable stage of erythroid differentiation.

A novel approach for the topographical localization of glycolipids on the cell surface

In this study we have developed a prototype system for distinguishing between the topographical distribution of glycolipids versus glycoproteins on the ultrastructural level. Direct modification of membrane-based sialic acids with biotin groups labels both glycolipids and glycoproteins. In this case, subsequent ultrastructural localization of biotinylated sites would not discern between these two classes of glycoconjugate in an unambiguous manner. When biotinylated cells are fixed prior to interaction with ferritin-conjugated avidin, the mean distance of marker molecules from the membrane bilayer is 8.0 nm. In contrast, if the cells are allowed to cap through the action of ferritin-avidin conjugates on unfixed cells, the average distance (13.0 nm) of the marker molecules appears even more distant from the membrane on the capped portion of the cell (uropod), whereas those on the head region are positioned in close proximity to the bilayer (3.7 nm). In order to exclusively label cell surface glycolipids on the ultrastructural level, bovine brain gangliosides were biotinylated in vitro and the haptenized gangliosides were incorporated into intact cells. In this case, marker molecules denoting the incorporated gangliosides were found in relatively close juxtaposition to the membrane surface, in a manner strikingly similar to the labeling pattern of the head region on capped cells. These results support the concept that, in the native state, the carbohydrate portion of glycolipids is positioned closer to the membrane bilayer than that of glycoproteins.