Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii.
Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii.
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DOI：
10.1083/jcb.124.5.795
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发表时间：
1994-03
期刊：
影响因子：
--
通讯作者：
Salisbury JL
中科院分区：
文献类型：
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作者：
Sanders MA;Salisbury JL
以前，我们报道了莱茵衣藻鞭毛切除是由一个主动过程介导的，在鞭毛-基体过渡区的选择位点，微管被切断（Sanders，M.一、和J.L.等。索尔兹伯里1989. 108：1751- 1760）。在鞭毛切除的时候，过渡区的星状纤维收缩并将轴丝的微管对向内移位。向内位移过程中产生的剪切力和扭转载荷导致微管在过渡区中心圆柱体远端立即切断。在这项研究中，我们使用了一个清洁剂提取的细胞模型的衣原体，允许直接实验访问的分子机制负责微管切断没有质膜的阻碍。我们提出了四个独立的实验证据的过渡区的中心蛋白为基础的星状纤维的鞭毛切除的过程中的基本参与：（a）洗涤剂提取的细胞模型切除他们的鞭毛响应升高，但生理，游离钙的水平。(b)用含有二价阳离子螯合剂EDTA的缓冲液提取细胞模型导致基于中心蛋白的纤维的分解和过渡区星状纤维结构的破坏。这种处理导致鞭毛切除能力的完全丧失。(c)三个单独的抗中心蛋白单克隆抗体制剂，定位于过渡区的星状纤维，特异性抑制星状纤维的收缩和阻断钙诱导的鞭毛切除，而对照抗体没有抑制作用。最后，（d）中心蛋白突变体vfl-2（Taillon，B.，S. Adler，J. Suhan，and J. Jarvik. 1992. J. Cell Biol.119：1613-1624）在活细胞中的pH休克或去污剂提取的细胞模型的钙处理后不能主动切除它们的鞭毛。两者合计，这些观察结果表明，基于中心蛋白的纤维收缩起着至关重要的作用，在微管切断时的鞭毛切除在衣原体。
Previously, we reported that flagellar excision in Chlamydomonas reinhardtii is mediated by an active process whereby microtubules are severed at select sites within the flagellar-basal body transition zone (Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751- 1760). At the time of flagellar excision, stellate fibers of the transition zone contract and displace the microtubule doublets of the axoneme inward. The resulting shear force and torsional load generated during inward displacement leads to microtubule severing immediately distal to the central cylinder of the transition zone. In this study, we have used a detergent-extracted cell model of Chlamydomonas that allows direct experimental access to the molecular machinery responsible for microtubule severing without the impediment of the plasma membrane. We present four independent lines of experimental evidence for the essential involvement of centrin-based stellate fibers of the transition zone in the process of flagellar excision: (a) Detergent-extracted cell models excise their flagella in response to elevated, yet physiological, levels of free calcium. (b) Extraction of cell models with buffers containing the divalent cation chelator EDTA leads to the disassembly of centrin-based fibers and to the disruption of transition zone stellate fiber structure. This treatment results in a complete loss of flagellar excision competence. (c) Three separate anti-centrin monoclonal antibody preparations, which localize to the stellate fibers of the transition zone, specifically inhibit contraction of the stellate fibers and block calcium-induced flagellar excision, while control antibodies have no inhibitory effect. Finally, (d) cells of the centrin mutant vfl-2 (Taillon, B., S. Adler, J. Suhan, and J. Jarvik. 1992. J. Cell Biol. 119:1613-1624) fail to actively excise their flagella following pH shock in living cells or calcium treatment of detergent-extracted cell models. Taken together, these observations demonstrate that centrin-based fiber contraction plays a fundamental role in microtubule severing at the time of flagellar excision in Chlamydomonas.