Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules.
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 suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 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 (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 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 mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.
Kowit
J D
JD
Emmanuel College, Boston, MA 02215.
Linck
R W
RW
Kenney
D M
DM
D000470:Alkaloids / Q000494:pharmacology; D001792:Blood Platelets / Q000648:ultrastructure*; D002458:Cell Fractionation; D003598:Cytoskeletal Proteins / Q000502:physiology; D006801:Humans; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000502:physiology; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure*; D008970:Molecular Weight; D017239:Paclitaxel; D014357:Trypsin / Q000494:pharmacology
D000470:Alkaloids; D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; D014357:Trypsin; D017239:Paclitaxel