Preferential incorporation of tubulin into the junctional region of ciliary outer doublet microtubules: a model for treadmilling by lattice dislocation.
Even in the presence of colchicine or Taxol(R), sea urchin embryonic cilia undergo substantial steady-state turnover, with a rate of tubulin incorporation approaching half that seen in full regeneration [Stephens: Mol Biol Cell 8:2187-2198, 1997]. Preliminary experiments suggest that tubulin incorporates differentially into the most stable portion of the outer doublet, the junctional protofilaments [Stephens: Cell Struct Funct 24:413-418, 1999]. To explore this possibility further, embryos of the sea urchin Tripneustes gratilla, a ciliary length inducible system [Stephens: J Exp Zool 269:106-115, 1994a], were pulse labeled with (3)H leucine during steady-state turnover or induced elongation, followed by regeneration in the presence of unlabeled leucine. Cilia were isolated by hypertonic shock and fractionated into detergent-soluble membrane plus matrix, thermally-solubilized microtubule walls, and insoluble 9-fold symmetric remnants of A-B junctional protofilaments plus associated architectural elements. The fractions were resolved by SDS-PAGE and the specific activity of alpha-tubulin was determined. In cilia undergoing turnover or elongation during an isotope pulse, the specific activity of tubulin in the junctional region approximated that of precursor membrane plus matrix tubulin but surpassed that of the tubule wall by a factor of approximately 1.5. In cilia regenerated during an isotope chase, the specific activity of junctional tubulin exceeded that of both the membrane plus matrix and the tubule wall by a similar factor. These data indicate that tubulin is preferentially incorporated into junctional protofilaments during steady-state turnover, induced elongation and regeneration. A model for directional incorporation based on surface lattice discontinuities in the outer doublet is proposed.
Stephens
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Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. rstephen@bu.edu
D000818:Animals; D002923:Cilia / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents / Q000494:pharmacology; D008868:Microtubule Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D012394:Rosaniline Dyes / Q000494:pharmacology; D012617:Sea Urchins / Q000187:drug effects / Q000378:metabolism*; D013806:Theophylline / Q000494:pharmacology; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D007202:Indicators and Reagents; D008868:Microtubule Proteins; D012394:Rosaniline Dyes; D014404:Tubulin; C044103:tektins; C048139:Coomassie blue; D013806:Theophylline