Goldmann W H, Galneder R, Ludwig M, Xu W, Adamson E D, Wang N, Ezzell R M
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.
Exp Cell Res. 1998 Mar 15;239(2):235-42. doi: 10.1006/excr.1997.3915.
We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.
我们研究了一种小鼠F9胚胎癌细胞系，其中两个纽蛋白基因通过同源重组失活，该细胞系表现出黏附及铺展缺陷[科尔等人（1995年）《美国国家科学院院刊》92, 9161 - 9165]。我们使用磁力计和RGD包被的磁性微珠，测量了纽蛋白缺失和替代对通过整合素传递机械力的局部影响。与野生型细胞相比，缺乏纽蛋白的F9Vin(-/-)细胞在相对硬度上有21%的差异。在向F9Vin(-/-)细胞中转染并组成型表达越来越多的纽蛋白后，这一差异恢复到接近野生型水平。相反，在F9Vin(-/-)细胞中转染缺少氨基酸1 - 288（包含与踝蛋白和α - 辅肌动蛋白结合位点）的纽蛋白构建体，或用酪氨酸替代苯丙氨酸（磷酸化位点，氨基酸822），导致硬度部分恢复。通过原子力显微镜以128×128（n = 16,384）次力扫描绘制整个F9细胞的相对弹性图谱，我们观察到与磁力计测量结果具有相关性。这些发现表明，纽蛋白可能通过稳定黏着斑并传递驱动细胞骨架重塑的机械应力来促进细胞黏附和铺展，从而影响细胞的弹性特性。
