Cramer L P
MRC-Laboratory Molecular Cell Biology University College London Gower Street, WC1E 6BT, The Randall Institute Kings College London, WC2B 5RL, UK.
Curr Biol. 1999 Oct 7;9(19):1095-105. doi: 10.1016/s0960-9822(99)80478-3.
In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer.
In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide.
Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.
在运动细胞中，片状伪足（一种细胞边缘类型）的突出需要肌动蛋白单体在片状伪足尖端组装成肌动蛋白丝。肌动蛋白丝解聚在这个过程中的重要性尚未得到充分理解，本文使用肌动蛋白药物茉莉酸内酯进行评估，该药物具有两种已知活性——抑制丝解聚和诱导肌动蛋白聚合物增加。
在细胞中发现茉莉酸内酯的两种活性是可分离的；1微摩尔茉莉酸内酯可渗透细胞，结合细胞丝状肌动蛋白（F-肌动蛋白）并在3.5分钟内抑制丝解聚，但直到处理60分钟后才检测到肌动蛋白聚合物有显著增加。在活的、透化的细胞中，茉莉酸内酯不抑制从提供的纯化肌动蛋白单体组装丝。在用1微摩尔茉莉酸内酯处理的1 - 5分钟内，迁移的鸡成纤维细胞中的片状伪足突出被阻断，而肌动蛋白组织没有任何扰动。在未迁移的鸡成纤维细胞中，茉莉酸内酯诱导的片状伪足突出抑制开始有延迟，在此期间片状伪足长度呈线性增加而突出速率没有增加。在用1微摩尔茉莉酸内酯处理3分钟内，感染的PtK2细胞中李斯特菌的运动性降低了2.3倍。
在迁移的鸡成纤维细胞中，肌动蛋白丝解聚与片状伪足突出紧密相关，在PtK2细胞中与李斯特菌的运动性紧密相关。对这些数据的一种简单解释是，正在进行的肌动蛋白丝组装优先使用来自丝解聚的游离肌动蛋白单体，而不是储存的单体。
