Immunocytochemical localization of coated vesicle protein in rodent nervous system.

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.

Ganglioside patterns and cholera toxin--peroxidase labeling of aggregating cells from the chick optic tectum.

2The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. GD3 and GD1b were the predominant gangliosides early in development, while GD1a and several other multisialogangliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin--peroxidase binding site GM1 showed conjugate binding correlates with increasing levels of GM1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of GM1 in the synaptic cleft region.

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum.

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0--4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1--2 DIV. By 7 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8--10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.

Lectin cytochemistry of carbohydrates on cell membranes of rat cerebellum.

Wheat germ agglutinin (WGA), Ricinis communis agglutinin (RCA) and Lens culinaris (LC) lectins have been used to characterize carbohydrates of neuronal membrane systems in rat and chick cerebellum. WGA and RCA both label Golgi membrane cisternae on the side of the membrane facing the cisternal space but they do not label other elements of the granular and agranular endoplasmic reticulum (ER). WGA labels the plasma membrane generally and WGA binding sites are concentrated within the synaptic cleft and at specialized sites along the axolemma at the node of Ranvier. RCA labelling of plasma membranes is sparse. Neuraminidase treatment of tissue slices prior to lectin cytochemistry does not alter the Golgi membrane distribution of WGA and RCA binding sites and other ER membranes remain unlabelled. The plasma membrane staining with WGA is decreased and that with RCA is increased after neuraminidase pretreatment. The pattern of lectin labelling with LC resembles that seen with concanavalin A (con A) in that all elements of the ER within cell bodies label on the side of the membrane facing the cisternal space. A major difference when compared to con A labelling is that the hypolemmal cisternae lying adjacent to the plasma membrane of Purkinje cell axons and dendrites do not label with LC. Collectively, these results suggest that elements of the ER of Purkinje cells are heterogeneous with respect to their lectin binding properties in a manner which is consistent with the formation of more complex oligosaccharides on membranes of the cell periphery.

Structure and function of the rubella virus proteins.

Rubella virus strain HPV-77 contains three structural polypeptides. The nucleocapsid is constructed with the C polypeptide chain, which has a molecular weight of 30,000. The envelope proteins are constructed with two glycopolypeptides; the E1 glycopolypeptide has a molecular weight of 63,000, and the E2 glycopolypeptide has a molecular weight of 45,000-48,000. The nucleocapsid capsomere is approximately 6.2S, has a molecular weight of 130,000, and consists of two disulfide-linked dimers of the C polypeptide. The E1 and E2 glycopolypeptides are associated, but the size and structure of the spike protein have not been determined. Strain-specific antigens were detected within the E2 glycopolypeptide in all of the strains tested. In chronologic serum samples, the titers of antibody to the E2 and C polypeptides were the first to decline. Hybridomas were produced that exhibited hemagglutination-inhibiting and neutralizing activities; however, these functions did not always correlate. Both the hemagglutination-inhibiting and neutralizing antibodies reacted with the E1 glycopolypeptide.