Identification and spatial arrangement of high molecular weight proteins (Mr 300 000-330 000) co-assembling with microtubules from a cultured cell line (rat glioma C6)

Eur J Cell Biol. 1985 Jul;38(1):149-56.

Abstract

The distribution of three high molecular weight proteins, MAP-1 (Mr 330 000), MAP-2 (Mr 300 000) and plectin (Mr 300 000) in various fractions obtained in cycles of temperature-dependent polymerization/depolymerization of microtubules from rat glioma C6 cells was studied. Using gel electrophoresis and immunoautoradiography/immunoblotting all three proteins were found to codistribute only partially with tubulin because considerable parts remained in the cold-insoluble fractions. Moreover, the proteins, particularly MAPs, were proteolytically degraded during cycling. By contrast, when microtubules were polymerized with taxol after isotonic cell lysis a considerable enrichment of MAP-1 and MAP-2 was achieved; again, plectin co-distributed only partially. In this procedure too, MAPs, especially MAP-2, were found to be highly subject to proteolysis, unless free Ca2+-ions were rigorously avoided. Proteolytic fragments generated from MAP-2 were of similar size independent of whether temperature- or taxol-dependent polymerization procedures were used, suggesting the occurrence of a MAP-2-specific protease. When the spatial arrangement of the high Mr proteins on taxol-polymerized C6 cell microtubules was directly visualized using gold-immunoelectron microscopy, a periodical, apparently helical, decoration of microtubules was found for MAP-1 and MAP-2; plectin was irregularly arrayed. A predominantly helical arrangement of both MAPs was demonstrated also for microtubules reconstituted from mammalian brain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaloids
  • Animals
  • Calcium / metabolism
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Glioma / ultrastructure
  • Intermediate Filament Proteins*
  • Microscopy, Electron
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules / metabolism*
  • Molecular Weight
  • Paclitaxel
  • Plectin
  • Protein Binding
  • Proteins / metabolism*
  • Rats

Substances

  • Alkaloids
  • Intermediate Filament Proteins
  • Microtubule-Associated Proteins
  • Plec protein, rat
  • Plectin
  • Proteins
  • Paclitaxel
  • Calcium