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).
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.
Koszka
C
C
Leichtfried
F E
FE
Wiche
G
G
D000470:Alkaloids; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma / Q000648:ultrastructure; D007381:Intermediate Filament Proteins; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D017239:Paclitaxel; D051190:Plectin; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D000470:Alkaloids; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C495334:Plec protein, rat; D051190:Plectin; D011506:Proteins; D017239:Paclitaxel; D002118:Calcium