Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Abstract

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the “hoop” shape of MB cytoskeletons in unfixed suspension where the microtubule coils had a mean diameter of 2.87 μm (± 0.18 μm, SD). Microtubules were uncoiled by brief exposure to trypsin (2 mg/μg protein) or by NaCl (154–600 mM) but not by DNase I, which removed ∼40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (α- and β-tubulin, IEF-51K, or actin) were found. However, a number (> 10) of minor polypeptides, each <5% of total cytoskeletal protein and with an Mr ranging from 80,000⪰ 260,000, were decreased in “uncoiled” MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanisms(s) involved in maintaining the microtubule coil of the platelet marginal band.