Evidence for a non-tubulin spindle matrix and for spindle components immunologically related to tektin filaments.
Tektins were originally described as a set of three filamentous proteins (tektin A, B and C) associated with the walls of axonemal microtubules of sea urchin sperm. Using affinity-purified polyclonal antibodies raised against tektins of two sea urchin species, Lytechinus pictus and Strongylocentrotus purpuratus, we looked for tektin-like components in microtubule systems other than axonemes. By immunofluorescence microscopy we observed labeling of meiotic spindles in eggs of the surf clam Spisula solidissima and in several mammalian cell lines. In Spisula eggs the tektin-like antigens were still associated with the spindles after about 95% of the tubulin had been removed via a calcium/cold treatment. In pig kidney epithelial cells the tektin-like antigen appeared to be associated with bundles of calcium-stable spindle microtubules. By SDS-PAGE immunoblot the affinity-purified anti-tektins recognized several polypeptides in tubulin-depleted spindle remnants of Spisula eggs: A approximately 52 kDa, 1 M KCl-resistant component was identified by the antibody raised against tektin C from S. purpuratus, a approximately 48 kDa component was recognized by the antibody specific for tektin A from L. pictus, and three polypeptide bands (approximately 64 kDa, approximately 100 kDa and greater than 200 kDa) were detected by the antibody specific for tektin C from L. pictus. Only the latter antibody, however, stained Spisula spindles by immunofluorescence microscopy. We further report that the sensitivity of antibody recognition of proteins on immunoblots is dependent on the purity of sodium dodecyl sulfate.
Steffen
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University of Minnesota, Department of Cell Biology and Neuroanatomy, Minneapolis 55455.
Linck
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RW
D000818:Animals; D049872:Bivalvia / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008297:Male; D016253:Microscopy, Immunoelectron; D008868:Microtubule Proteins / Q000032:analysis* / Q000276:immunology; D012617:Sea Urchins / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry / Q000648:ultrastructure; D008941:Spindle Apparatus / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D013552:Swine; D014404:Tubulin / Q000032:analysis
D008868:Microtubule Proteins; D014404:Tubulin; C044103:tektins; D002118:Calcium