Chan A Y, Raft S, Bailly M, Wyckoff J B, Segall J E, Condeelis J S
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
J Cell Sci. 1998 Jan;111 ( Pt 2):199-211. doi: 10.1242/jcs.111.2.199.
Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/-0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.
用表皮生长因子（EGF）刺激转移性MTLn3细胞会导致片状伪足迅速伸展，片状伪足的前沿含有一个F-肌动蛋白区域。为了确定F-肌动蛋白在前沿积累的机制及其与EGF刺激下片状伪足伸展的关系，我们研究了MTLn3细胞中EGF诱导的肌动蛋白成核活性的动力学和位置，并通过测量MTLn3细胞在EGF刺激下肌动蛋白丝的尖端和倒刺端的变化来表征前沿的肌动蛋白动力学。这项研究的主要结果是，用EGF刺激MTLn3细胞会导致肌动蛋白成核活性短暂增加，这是由于在伸展的片状伪足前沿非常靠近处出现了游离的倒刺端。此外，细胞松弛素D会使EGF刺激的细胞中总F-肌动蛋白含量显著降低，这表明EGF既刺激了肌动蛋白的聚合又刺激了其解聚。EGF刺激的细胞对罗丹明标记的肌动蛋白的尖端掺入比对照细胞高2.12±0.47倍。由于EGF刺激会导致同一位置的罗丹明标记的肌动蛋白在倒刺端和尖端的掺入都增加，因此EGF刺激的成核位点更可能是由于预先存在的细丝的切断或前沿细丝的从头成核，从而产生新的倒刺端和尖端。MTLn3细胞中EGF诱导的肌动蛋白成核活性的时间和位置可以解释观察到的F-肌动蛋白在前沿的积累，并证明这个富含F-肌动蛋白的区域是刺激后主要的肌动蛋白聚合区域。
