Wargo Matthew J, Dymek Erin E, Smith Elizabeth F
Dartmouth College, Department of Biological Sciences, 301 Gilman Hall, Hanover, NH 03755, USA.
J Cell Sci. 2005 Oct 15;118(Pt 20):4655-65. doi: 10.1242/jcs.02585. Epub 2005 Sep 27.
Studies of flagellar motility in Chlamydomonas mutants lacking specific central apparatus components have supported the hypothesis that the inherent asymmetry of this structure provides important spatial cues for asymmetric regulation of dynein activity. These studies have also suggested that specific projections associated with the C1 and C2 central tubules make unique contributions to modulating motility; yet, we still do not know the identities of most polypeptides associated with the central tubules. To identify components of the C1a projection, we took an immunoprecipitation approach using antibodies generated against PF6. The pf6 mutant lacks the C1a projection and possesses flagella that only twitch; calcium-induced modulation of dynein activity on specific doublet microtubules is also defective in pf6 axonemes. Our antibodies specifically precipitated five polypeptides in addition to PF6. Using mass spectrometry, we determined the amino acid identities of these five polypeptides. Most notably, the PF6-containing complex includes calmodulin. Using antibodies generated against each precipitated polypeptide, we confirmed that these polypeptides comprise a single complex with PF6, and we identified specific binding partners for each member of the complex. The finding of a calmodulin-containing complex as an asymmetrically assembled component of the central apparatus implicates the central apparatus in calcium modulation of flagellar waveform.
该结构固有的不对称性为动力蛋白活性的不对称调节提供了重要的空间线索。这些研究还表明,与C1和C2中央微管相关的特定突起对调节运动性有独特贡献;然而,我们仍然不知道与中央微管相关的大多数多肽的身份。为了鉴定C1a突起的成分,我们采用了免疫沉淀方法,使用针对PF6产生的抗体。pf6突变体缺乏C1a突起,其鞭毛只能抽搐;pf6轴丝中钙诱导的特定双联体微管上动力蛋白活性的调节也存在缺陷。我们的抗体除了沉淀PF6外,还特异性沉淀了五种多肽。通过质谱分析,我们确定了这五种多肽的氨基酸身份。最值得注意的是,含PF6的复合物包括钙调蛋白。使用针对每种沉淀多肽产生的抗体,我们证实这些多肽与PF6组成一个单一复合物,并且我们鉴定了该复合物每个成员的特定结合伴侣。发现含钙调蛋白的复合物是中央装置的不对称组装成分,这表明中央装置参与了鞭毛波形的钙调节。