Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle.

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I-band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.

A new antibody label 5-10 times smaller than previous ones

By covalently coupling an undecagold complex to an Fab’ fragment, the smallest gold-conjugated antibody label yet devised has been synthesized. Its overall dimension is 50 x 30 x 20 Å and when bound to an antigen, the gold cluster is 40-50 Å from the antigen surface. The preparation procedure does not alter the activity and specificity of the antibody since coupling of the gold cluster is at a crystallographically known position at the opposite end of the Fab’ fragment from the antigen combining site.One significance of this new label is that with a smaller E.M. antibody label, higher resolution of labeling is possible. In fact, if monoclonals are used, specific sites on a single macromolecule, or subunits of a protein complex could be mapped.

Receptor patching and capping of platelet membranes induced by monoclonal antibodies

Redistribution of glycoproteins (GP) Ib, glycocalicin, IIb, and IIIa on the surface of human platelets in response to stimulation with corresponding monoclonal antibodies (MoAb) and a polyclonal antiglycocalicin antibody was studied by immunofluorescence, immunoelectron microscopy, and a quantitative radioimmune assay. Immobilization of the antigens by prefixation with formaldehyde showed a uniform distribution over the surface of the platelet. Incubation of unfixed platelets with specific MoAb against various epitopes on GPIIb and/or IIIa resulted in a time-dependent patching, subsequent capping, and after prolonged exposure to the antibody/label complex, internalization of the complex, possibly by endocytosis. In contrast, GPIb could not be shown to cap. From these results we conclude that platelet GPIIb and/or IIIa undergo spatial rearrangement in a manner analogous to that observed in lymphocytes, whereas GPIb does not. Since both GPIb and GPIIb and/or IIIa seem to be transmembraneous GP associated with the cytoskeleton, a special, though unidentified, role of GPIIb/IIIa in the induction of lateral membrane mobility is postulated.

Receptor patching and capping of platelet membranes induced by monoclonal antibodies

Redistribution of glycoproteins (GP) Ib, glycocalicin, IIb, and IIIa on the surface of human platelets in response to stimulation with corresponding monoclonal antibodies (MoAb) and a polyclonal antiglycocalicin antibody was studied by immunofluorescence, immunoelectron microscopy, and a quantitative radioimmune assay. Immobilization of the antigens by prefixation with formaldehyde showed a uniform distribution over the surface of the platelet. Incubation of unfixed platelets with specific MoAb against various epitopes on GPIIb and/or IIIa resulted in a time-dependent patching, subsequent capping, and after prolonged exposure to the antibody/label complex, internalization of the complex, possibly by endocytosis. In contrast, GPIb could not be shown to cap. From these results we conclude that platelet GPIIb and/or IIIa undergo spatial rearrangement in a manner analogous to that observed in lymphocytes, whereas GPIb does not. Since both GPIb and GPIIb and/or IIIa seem to be transmembraneous GP associated with the cytoskeleton, a special, though unidentified, role of GPIIb/IIIa in the induction of lateral membrane mobility is postulated.