Retention of ciliary ninefold structure after removal of microtubules.
When axonemes of isolated gill cilia from the bay scallop Aequipecten irradians are heated at 45 degrees C for a minimum of 8 min in a 10 mM-Tris-HCl (pH 8), 1 mM-EDTA solution, nearly 80% of the tubulin is solubilized but most minor structural proteins are retained in a ninefold symmetrical configuration. This remnant consists of the junctional protofilaments, derived from outer doublet tubules, interconnected by nexin linkages, with radial spoke components still directed inwards. The remnant is of the same length as the original cilium, with the junctional protofilaments attached at the distal end to the ciliary tip and at the proximal end to the basal plate. Virtually identical fractionations can be achieved with blastula cilia isolated from both arctic and tropical sea-urchin embryos. The remnant is resistant to salt up to at least 1 M concentration, judged by the constancy of protein composition. Immunoblotting with antibodies against sea-urchin sperm flagellar tektins indicates that the tektins remain within the ciliary remnant, supporting their location within the junctional protofilament domain. The fractionation is inhibited by low pH, by magnesium or calcium ions in the millimolar range, and by monovalent ions at 10-fold higher concentrations. About a quarter of the total ciliary calmodulin is bound to the axoneme at micromolar calcium levels but most is released upon thermal fractionation. Polymerization of tubulin in the presence of the remnant results in singlet microtubules, separate from the remnant proper, suggesting that doublet formation may require coordinate co-assembly of tubulin with skeletal proteins. These observations demonstrate the existence of a fibrous skeleton in the axoneme, composed largely of ciliary tektins, nexin linkages, and other structural proteins.
Stephens
R E
RE
Marine Biological Laboratory, Woods Hole, MA 02543.
Oleszko-Szuts
S
S
Linck
R W
RW
D000818:Animals; D002147:Calmodulin / Q000032:analysis; D002923:Cilia / Q000648:ultrastructure*; D005880:Gills / Q000648:ultrastructure*; D008868:Microtubule Proteins / Q000032:analysis; D008870:Microtubules / Q000648:ultrastructure*; D008974:Mollusca / Q000033:anatomy & histology*; D012617:Sea Urchins / Q000033:anatomy & histology*
D002147:Calmodulin; D008868:Microtubule Proteins; C044103:tektins