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

Joel D. Kowit, Richard W. Linck, Dianne M. Kenney

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4 Scopus citations

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{succeeds above single-line equals sign} 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.

Original languageEnglish (US)
Pages (from-to)283-291
Number of pages9
JournalBiology of the Cell
Volume64
Issue number3
DOIs
StatePublished - 1988

Bibliographical note

Funding Information:
This investigationw as supported by USPHS grants HL24311 and HL29583 (to D.K.) from the National Heart and Lung Institute, by GM35648 (to R.W.L.) from the National Institute of General Medical Sciences, by a Cottreg College ScienceG rant (to J.D.K.) from The Researci~ Corporation, Providence, RI, and by National Science F~ndation Equip~e~t grant PCM 78-16158. The authors thank Lisa D. Weiss mtd Gerard A. Dillon for technical assistance.

Keywords

  • dark-field
  • marginal band
  • platelets

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