High-resolution backscatter electron detection of cell surface molecules on human platelets using the double-layer coating method and cryo field emission scanning electron microscopy.

A model system utilizing cryo scanning electron microscopy (SEM) for the detection of putative cell adhesion molecule(s) on the surface of human platelets is described. Plunge freezing was used for cryoimmobilization of unactivated and activated platelets after prefixation. Extracellular ice was removed by sublimation to expose the surface of the platelet membrane. Cryosamples were coated by the double-layer method, in which undirectional shadowing is performed at an angle of 45 degrees with 2 nm of platinum by thermal evaporation, followed by evaporation of 5 nm of carbon at an angle of 90 degrees for stabilization of the platinum film. The topography of the extracellular surface of the unstimulated platelet membrane was dominated by small spherical protrusions, while that of the activated platelet had not only similar spherical projections, but also possessed numerous rod-like protrusions, presumably representing the upregulation of the cell adhesion molecule, P-selectin, from intracellular a granules. These results clearly demonstrate that cryo field-emission SEM can detect molecular topography on the extracellular surface of cells consistent with the dimensions and shape of membrane cell adhesion molecules.

Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly.

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.

Concurrent effects of microsieve aspiration and the ionophore A23187 on the morphologic characteristics of the surface of normal erythrocytes.

Previous studies using the calcium ionophore A23187 have shown that pathologic changes in erythrocyte shape and deformability can be induced by modest increases in intracellular calcium with the concomitant depletion of cellular adenosine triphosphate. Efforts to modify the cellular response to such ionophore-induced calcium loading by means of environmental and chemical manipulation of the erythrocyte membrane have suggested that the state of the membrane prior to calcium uptake influences the response of the cell. The present investigation used the microsieve aspiration technic to induce membrane tension mechanically prior to calcium loading with A23187, and thereby to evaluate the response of the membrane to another form of stress. Erthrocytes subjected to conditions of membrane tension in the microsieve were found to resist completely the A23187-calcium-induced conversion to echinocytes or spheroechinocytes. These results support the hypothesis that the state of the membrane before calcium loading has a marked influence on the response of the cell. This may be related to the mechanism underlying the development of irreversibly sickled cells in patients with sickle cell anemia.