Lefebvre P A, Nordstrom S A, Moulder J E, Rosenbaum J L
J Cell Biol. 1978 Jul;78(1):8-27. doi: 10.1083/jcb.78.1.8.
Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co-migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.
鞭毛脱落后,需要合成新的蛋白质以再生全长的衣藻鞭毛。利用蛋白质合成基础水平较低的配子,已能够在全细胞中标记并检测许多鞭毛蛋白的合成。在配子细胞质中可检测到由鞭毛脱落诱导的微管蛋白、动力蛋白、鞭毛膜蛋白以及至少20种其他与分离轴丝中的蛋白质共迁移的蛋白质的合成,并且可以研究每种蛋白质合成的起始和终止时间。刺激细胞启动鞭毛蛋白合成的信号的性质尚不清楚。鞭毛再生和随之而来的储备消耗对于鞭毛蛋白合成的起始或终止都不是必需的,因为阻断鞭毛再生的秋水仙碱不会改变鞭毛脱落后细胞质中合成的蛋白质模式或其合成时间。此外,在化学诱导细胞吸收其鞭毛后,鞭毛蛋白合成受到刺激,这表明鞭毛脱落本身对于刺激合成并非必需。已确定了通过从培养基中去除Ca++并提高K+或Na+浓度来诱导细胞吸收其鞭毛的方法。这种吸收是可逆的,并且在没有蛋白质合成的情况下,被吸收的鞭毛成分可重新用于组装鞭毛。本报告中使用这项新技术来研究鞭毛合成和组装的控制。