Lorenz Mike, DesMarais Vera, Macaluso Frank, Singer Robert H, Condeelis John
Albert Einstein College of Medicine, Department of Anatomy and Structural Biology, Bronx, NY 10461, USA.
Cell Motil Cytoskeleton. 2004 Apr;57(4):207-17. doi: 10.1002/cm.10171.
Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.
细胞运动性与富含丝状肌动蛋白（F-肌动蛋白）的突起延伸能力相关，并且依赖于游离的刺端作为新的肌动蛋白聚合位点。为了理解参与产生刺端过程的不同蛋白质（如丝切蛋白和Arp2/3）的功能和调控，已采用固定细胞方法来确定细胞中刺端的相对浓度。这些方法的主要缺点是细胞的透化处理和固定。在这项工作中，我们描述了一种新的活细胞延时显微镜检测方法，用于确定细胞刺激后刺端的增加，该方法不使用透化处理，并且提供了更好的时间分辨率。我们建立了一个在生理水平稳定表达绿色荧光蛋白-β-肌动蛋白（GFP-β-肌动蛋白）的转移性癌细胞系（MTLn3）。用表皮生长因子（EGF）刺激MTLn3细胞会导致快速且短暂的片状伪足突起，同时前沿肌动蛋白聚合增加，这在活细胞实验中可以观察到。通过测量前沿F-肌动蛋白随时间的增加，我们能够以高时间分辨率确定刺激后刺端的相对增加。F-肌动蛋白以及刺端浓度与该细胞系已发表的数据非常吻合。使用这种新开发的检测方法，在显微注射抗丝切蛋白功能阻断抗体后，记录到片状伪足延伸减少以及刺端大幅减少。该检测方法在测量动态丝状肌动蛋白群体快速变化的应用中具有很大潜力。
