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H Spiele

Publications and source records attributed to H Spiele.

15 recordsLinked to original sources
PubMed1987

Interaction of plasma membrane-associated filaments and H-2 histocompatibility antigens before and after induced patching and capping.

The interaction of H-2 antigens and plasma membrane-associated filaments was studied on dry-cleaved preparations of immunogold-labelled lymphoma cells. In prefixed cells, the plasma membrane-associated network was isotropic without any prevailing direction of the filaments, and the gold-labelled H-2 antigens were preferentially localized over or at a very short distance from membrane-associated filaments. Incubation of unfixed cells with anti-H-2 antibodies followed by fixation and incubation with anti-Ig, did not induce detectable redistribution of H-2 antigens or of the filament network. Notwithstanding this apparent absence of rearrangement of H-2 antigens and filaments, a detergent-resistant linkage to the cytoskeleton was induced. Before immune incubations, virtually all H-2 antigens were solubilized by extraction with Triton X-100, while after incubation with anti-H-2 antibodies about 50% of the H-2 antigens were linked to the Triton X-100-insoluble cytoskeleton. Sequential addition of anti-H-2 and anti-Ig antibodies to unfixed cells induced formation of patches and caps of H-2 antigens. Under these conditions, the majority of the H-2 antigens became linked to the detergent-resistant cytoskeleton. Redistribution into patches and caps was often accompanied by a local rearrangement of the isotropic network into bundles of parallel filaments immediately adjacent to the plasma membrane. Patches were seen to overly both isotropic networks and these parallel filaments. Large sheets of plasma membrane overlying parallel filaments were frequently devoid of gold-labelled H-2 antigens and coated pits, and thus most probably represented areas away from caps. This observation suggests that capping is accompanied by a rearrangement of filaments close to the membrane.

Animals↗
PubMed1985

Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.

Actin Cytoskeleton↗
PubMed1984

Brief extraction with detergent induces the appearance of many plasma membrane-associated microtubules in hepatocytic cells.

In cultured H35 hepatoma cells membrane-associated cortical networks have a microtrabecular appearance as revealed by dry-cleaving. Filaments having diameters of 15 nm can be readily distinguished within these networks and have not been described previously. Microtubules are seldom observed to be part of this structure. Extraction of cells with 0.1% Saponin in microtubule-stabilizing buffer produces holes in the membrane and reorganization of the networks resulting in the loss of microtrabecular structure, the loss of 15 nm filaments and the appearance of abundant membrane-associated microtubules (about 1.25 micron per micron2 substrate-adherent membrane). These observations were confirmed by immunolabelling experiments with affinity-purified anti-tubulin immunoglobulin G. By both fluorescence microscopy and electron microscopy it was shown that labelled tubulin in the cortical networks became organized into microtubules upon treatment with detergent. By determination of the microtubule density, expressed as micron microtubule per micron2 membrane, the effects of various conditions on microtubule occurrence were determined. The Saponin-induced appearance of microtubules in the membrane-associated network could be inhibited by: 1% and 2% glutaraldehyde, 0 degrees C, millimolar Ca2+, absence of Mg2+ (subsequent reversal of inhibition by addition of Mg2+ was shown), and 20 microM-nocodazole (but not 20 microM-colchicine). In addition to Saponin, extraction with 0.1% Nonidet P-40 or 0.1% Triton X-100 also resulted in microtubule-containing cortical networks. However, 0.1% Triton N-101 was not effective, although holes were produced in the plasma membrane. These data provide evidence suggesting rapid polymerization of membrane-associated microtubule protein rather than detergent-induced displacement or collapse of existing microtubules. The arguments for this hypothesis and its implications are discussed.

Animals↗
PubMed1983

Plasma membrane-associate filament systems in cultured cells visualized by dry-cleaving.

Substrate-attached critical-point-dried cells cleave along the level of the substrate-adherent membrane if removed by means of adhesive tape. The remaining membrane fragments on grids can be visualized three-dimensionally by means of stereo transmission electron microscopy. Attachment of cells may be achieved by active spreading of the cell, or artificially by poly-L-lysine adherence of prefixed cells. In 11 different cell types a filamentous network appears to remain associated with the cytoplasmic face of the membrane. In one hepatoma cell type virtually no filamentous network could be detected. Two general network morphologies are described: the hepatocytic network and the lymphoid network. Since no correspondence could be found between cytoplasmic structure and the structure of the membrane-associated network, and since cells generally cleave along the level of this network, excluding cell organelles, we conclude that it comprises a distinct structural system, analogous to the membrane skeleton of the red cell membrane.

Animals↗
PubMed1981

Effect of vinblastine and cytochalasin B on the cytoskeletal domains in 3T3 cells.

Normal 3T3 cells were exposed to vinblastine and cytochalasin B in an attempt to correlate the morphological changes of the cell surface as seen in the scanning electron microscope with ultrastructural changes of the cytoskeletal elements as seen in critical-point-dried cells in the transmission electron microscope. Special attention was given to the changes in the cytoplasmic domains distinguished in a previous paper. Cytochalasin B primarily affects the ultrastructure of the cytocortical domain by inducing the formation of condensation foci on the cytoplasmic material. Vinblastine not only induces the depolymerization of microtubules and the perinuclear concentration of intermediate filaments, but it also causes the disappearance of stress fibres from the cortical cytoplasm and the widening of the cytocortex at the expense of the endoplasmic domain. These results support the hypothesis that the differentiation in ultrastructural domains is dependent on the spreading of the cells and their adhesion to substrate.

Animals↗
PubMed1980

Different cytoskeletal domains in murine fibroblasts.

The morphology of the cytoskeleton of normal 3T3 fibroblasts was studied in the electron microscope on embedded and sectioned cells as well as on critical-point-dried whole mounts. Four ultrastructurally different cytoplasmic domains could be distinguished within every 3T3 cell, namely: Cytocortex, ruffle area, endoplasm, and stress fibre. However, many transitions between the different domains were also found. In addition, 2 other domains were occasionally encountered in the same cells. Based on the knowledge that actin is present in relatively high concentration throughout non-muscle cells and that it can, under different physiological conditions, assume different morphological appearances, an attempt has been made to formulate a hypothesis that links the ultrastructural domains to different cellular functions. Many experimental approaches are available to test the hypothesis and at the same time increase our knowledge of the cytoskeleton of 3T3 cells.

Actins↗
PubMed1978

Preservation of cytoskeletal elements for electron microscopy.

Attached murine fibroblasts were subjected to different fixation and dehydration procedures in order to study the preservation of cytoskeletal elements. The organization of the microtubular and microfilamentous system was identical whether or not post-fixed with osmic acid after glutaraldehyde fixation. Critical point drying did not preserve structures absent in conventionally dehydrated and embedded cells.

Animals↗