"Bundle blot" purification and initial protein characterization of hair cell stereocilia.
Stereocilia were isolated from bullfrog (Rana catesbeiana) saccular hair cells by nitrocellulose adhesion. The high purity and high yield of the preparation were demonstrated by microscopy. SDS/PAGE of stereociliary proteins resolved 12-15 major bands. Actin, previously identified as a component of the stereociliary core, was identified in purified stereocilia as a band comigrating with authentic actin and by phalloidin labeling of intact isolated stereocilia. Fimbrin was identified in immunoblots of purified stereocilia. The most abundant other proteins migrated at 11, 14, 16-19, 27, and 36 kDa. Demembranated stereociliary cores consisted primarily of protein bands corresponding to actin and fimbrin and several proteins ranging from 43 to 63 kDa. Because the adaptation mechanism in hair cells is calcium-sensitive and seems localized to stereocilia, we sought evidence for calcium-binding proteins in stereocilia. Calmodulin and calbindin antibodies labeled stereocilia in intact cells. A protein band in purified stereocilia exhibited a Ca2+-dependent shift in electrophoretic mobility identical to that of authentic calmodulin, and the 27-kDa band may represent calbindin. These biochemical data demonstrate that stereocilia consist of a relatively small set of proteins. Most of these, including those involved in transduction and adaptation, are as yet uncharacterized. The availability of purified stereocilia should prove useful in further studies of structure-function relationships in these mechanically sensitive organelles.
Shepherd
G M
GM
Howard Hughes Medical Institute, Massachusetts General Hospital, Boston 02114.
Barres
B A
BA
Corey
D P
DP
D000199:Actins / Q000032:analysis*; D000818:Animals; D002923:Cilia / Q000648:ultrastructure*; D004847:Epithelial Cells; D004848:Epithelium / Q000648:ultrastructure; D006198:Hair Cells, Auditory / Q000648:ultrastructure*; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D011892:Rana catesbeiana
D000199:Actins; D008565:Membrane Proteins