Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes.
Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.
Nakagawa
Y
Y
Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Yamane
Y
Y
Okanoue
T
T
Tsukita
S
S
Tsukita
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001648:Bile Canaliculi / Q000737:chemistry; D002478:Cells, Cultured; D002502:Centrioles / Q000737:chemistry*; D018385:Centrosome / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016193:G1 Phase; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D013094:Spermatozoa / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D006360:Heat-Shock Proteins; C490107:ODF2 protein, human; C490106:Odf2 protein, mouse; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase