Pendleton A, Koffer A
Physiology Department, University College London, University Street, London, United Kingdom.
Cell Motil Cytoskeleton. 2001 Jan;48(1):37-51. doi: 10.1002/1097-0169(200101)48:1<37::AID-CM4>3.0.CO;2-0.
To investigate the role of the actin cytoskeleton in exocytosis, we have tested the effects of latrunculin B, a microfilament-disrupting drug, on secretion from intact and permeabilised rat peritoneal mast cells. The toxin strongly inhibited secretion from intact cells (attached or in suspension) responding to a polybasic agonist, compound 48/80. However, this effect was revealed only after a profound depletion of actin filaments. This was achieved by a long (1 h) exposure of cells to the drug before activation, together with its presence during activation. Maximal inhibition of secretion by such treatment was 85% at 40 microgram/ml latrunculin B. These results indicate that minimal actin structures are essential for the exocytotic response. In contrast, stimulus-induced cell spreading was prevented by latrunculin (5 microgram/ml) applied either before or after activation. The effects of the toxin on intact cells were fully reversible. The responses of permeabilised cells were affected differentially: secretion induced by calcium was more sensitive to latrunculin than that induced by GTP-gamma-S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton than the response induced by GTP-gamma-S. Again, maximal inhibitory effects (approximately 65 and 25% at 40 microgram/ml) were observed only when cells were exposed to the toxin both before and after permeabilisation. Since the permeabilised cells system focuses on the final steps of exocytosis, the incomplete inhibition suggests that actin plays a modulatory rather than a central role at this stage.
为了研究肌动蛋白细胞骨架在胞吐作用中的作用，我们测试了微丝破坏药物拉春库林B对完整和通透的大鼠腹膜肥大细胞分泌的影响。该毒素强烈抑制完整细胞（贴壁或悬浮）对多碱性激动剂化合物48/80的分泌反应。然而，这种作用只有在肌动蛋白丝大量耗竭后才显现出来。这是通过在激活前将细胞长时间（1小时）暴露于该药物，并在激活过程中保持药物存在来实现的。在40微克/毫升拉春库林B作用下，这种处理对分泌的最大抑制率为85%。这些结果表明，最小的肌动蛋白结构对于胞吐反应至关重要。相反，无论在激活前还是激活后施加拉春库林（5微克/毫升），都能阻止刺激诱导的细胞铺展。毒素对完整细胞的作用是完全可逆的。通透细胞的反应受到不同影响：钙诱导的分泌比GTP-γ-S诱导的分泌对拉春库林更敏感。因此，钙反应比GTP-γ-S诱导的反应更依赖于肌动蛋白细胞骨架的完整性。同样，只有当细胞在通透前后都暴露于毒素时，才观察到最大抑制作用（在40微克/毫升时约为65%和25%）。由于通透细胞系统关注胞吐作用的最后步骤，不完全抑制表明肌动蛋白在这个阶段起调节作用而非核心作用。
