Turnover of tubulin in ciliary outer doublet microtubules.
Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling.
Stephens
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Department of Physiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA. rstephen@bu.edu
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002923:Cilia / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004398:Dyneins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008868:Microtubule Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D012617:Sea Urchins / Q000196:embryology / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D003902:Detergents; D008868:Microtubule Proteins; D011506:Proteins; D014404:Tubulin; C044103:tektins; D014316:Tritium; D004398:Dyneins; D007930:Leucine; D017239:Paclitaxel