Dentler W L, Adams C
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
J Cell Biol. 1992 Jun;117(6):1289-98. doi: 10.1083/jcb.117.6.1289.
To study the mechanisms responsible for the regulation of flagellar length, we examined the effects of colchicine and Cytochalasin D (CD) on the growth and maintenance of Chlamydomonas flagella on motile wild type cells as well as on pf 18 cells, whose flagella lack the central microtubules and are immobile. CD had no effect on the regeneration of flagella after deflagellation but it induced fully assembled flagella to shorten at an average rate of 0.03 microns-min. Cells remained fully motile in CD and even stubby flagella continued to move, indicating that flagellar shortening did not selectively disrupt machinery necessary for motility. To observe the effects of the drug on individual cells, pf 18 cells were treated with CD and flagella on cells were monitored by direct observation over a 5-hour period. Flagella on control pf 18 cells maintained their initial lengths throughout the experiment but flagella on CD-treated cells exhibited periods of elongation, shortening, and regrowth suggestive of the dynamic behavior of cytoplasmic microtubules observed in vitro and in vitro. Cells behaved individually, with no two cells exhibiting the same flagellar behavior at any given time although both flagella on any single cell behaved identically. The rate of drug-induced flagellar shortening and elongation in pf 18 cells varied from 0.08 to 0.17 microns-min-1, with each event occurring over 10-60-min periods. Addition of colchicine to wild type and pf 18 cells induced flagella to shorten at an average rate of 0.06 microns-min-1 until the flagella reached an average of 73% of their initial length, after which they exhibited no further shortening or elongation. Cells treated with colchicine and CD exhibited nearly complete flagellar resorption, with little variation in flagellar length among cells. The effects of these drugs were reversible and flagella grew to normal stable lengths after drug removal. Taken together, these results show that the distal half to one-third of the Chlamydomonas flagellum is relatively unstable in the presence of colchicine but that the proximal half to two-thirds of the flagellum is stable to this drug. In contrast to colchicine, CD can induce nearly complete flagellar microtubule disassembly as well as flagellar assembly. Flagellar microtubules must, therefore, be inherently unstable, and flagellar length is stabilized by factors that are sensitive, either directly or indirectly, to the effects of CD.
为了研究负责鞭毛长度调节的机制，我们检测了秋水仙碱和细胞松弛素D（CD）对运动型野生型细胞以及pf 18细胞（其鞭毛缺乏中央微管且不能运动）的衣藻鞭毛生长和维持的影响。去鞭毛后，CD对鞭毛再生没有影响，但它会诱导完全组装好的鞭毛以平均每分钟0.03微米的速度缩短。细胞在CD中仍能完全运动，即使是短粗的鞭毛也继续摆动，这表明鞭毛缩短并没有选择性地破坏运动所需的机制。为了观察药物对单个细胞的影响，用CD处理pf 18细胞，并在5小时内通过直接观察监测细胞上的鞭毛。对照pf 18细胞上的鞭毛在整个实验过程中保持其初始长度，但用CD处理的细胞上的鞭毛表现出伸长、缩短和再生长的阶段，这暗示了在体外和体内观察到的细胞质微管的动态行为。细胞表现独立，在任何给定时间没有两个细胞表现出相同的鞭毛行为，尽管任何单个细胞上的两条鞭毛行为相同。pf 18细胞中药物诱导的鞭毛缩短和伸长速率在每分钟0.08至0.17微米之间，每个事件发生在10 - 60分钟的时间段内。向野生型和pf 18细胞中添加秋水仙碱会诱导鞭毛以平均每分钟0.06微米的速度缩短，直到鞭毛达到其初始长度的平均73%，此后它们不再进一步缩短或伸长。用秋水仙碱和CD处理的细胞表现出几乎完全的鞭毛吸收，细胞间鞭毛长度变化很小。这些药物的作用是可逆的，去除药物后鞭毛生长到正常的稳定长度。综上所述，这些结果表明，在秋水仙碱存在的情况下，衣藻鞭毛的远端二分之一到三分之一相对不稳定，但鞭毛的近端二分之一到三分之二对这种药物是稳定的。与秋水仙碱相反，CD可以诱导几乎完全的鞭毛微管解聚以及鞭毛组装。因此，鞭毛微管本质上一定是不稳定的，鞭毛长度由直接或间接对CD的作用敏感的因素稳定。
