Janmey P A, Hvidt S, Käs J, Lerche D, Maggs A, Sackmann E, Schliwa M, Stossel T P
Experimental Medicine Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
J Biol Chem. 1994 Dec 23;269(51):32503-13.
To address large discrepancies reported in the literature, the viscoelastic properties of gels formed by purified actin filaments have been measured by five different techniques and five different instruments using actin preparations purified separately in four different laboratories. These measurements consistently showed that the elastic shear modulus of 2 mg/ml F-actin is on the order of several hundred pascals, and depends very strongly on the length of the filaments and on the history of the sample prior to measurement. Shortening of actin filaments with gelsolin and mechanical perturbations reduce the shear modulus to low values identical to some reported in the literature, indicating that such perturbations account for low shear moduli and poor responsiveness to filament modifying treatments reported previously. The structures of individual actin filaments within gels very similar or identical to those studied rheometrically were also examined by dynamic light scattering and fluorescence microscopy. Dynamic light scattering data were analyzed by a new method to confirm that actin filaments have no stable associations with each other and fluctuate in solution at a rate governed by the filament bending modulus or persistence length, determined to be approximately 10 microns. Fluorescence microscopy confirmed that applying even small shear stresses to F-actin can orient and rupture the filaments, and that in a minimally perturbed viscoelastic gel, long actin filaments are free to diffuse within a limit of constraints formed by their neighbors. These findings confirm that relatively isotropic F-actin networks are sufficiently strong to stabilize cells.
为了解决文献中报道的巨大差异，使用在四个不同实验室分别纯化的肌动蛋白制剂，通过五种不同技术和五种不同仪器测量了由纯化的肌动蛋白丝形成的凝胶的粘弹性特性。这些测量结果一致表明，2mg/ml F-肌动蛋白的弹性剪切模量约为几百帕斯卡，并且非常强烈地依赖于丝的长度以及测量前样品的历史。用凝溶胶蛋白缩短肌动蛋白丝和机械扰动会将剪切模量降低到与文献中报道的一些值相同的低值，这表明这种扰动导致了先前报道的低剪切模量和对丝修饰处理的低响应性。还通过动态光散射和荧光显微镜检查了凝胶中与通过流变学研究的那些非常相似或相同的单个肌动蛋白丝的结构。通过一种新方法分析动态光散射数据，以确认肌动蛋白丝彼此之间没有稳定的缔合，并且在溶液中以由丝弯曲模量或持久长度决定的速率波动，持久长度确定约为10微米。荧光显微镜证实，即使对F-肌动蛋白施加很小的剪切应力也会使丝定向并断裂，并且在最小扰动的粘弹性凝胶中，长肌动蛋白丝在其相邻丝形成的约束范围内可以自由扩散。这些发现证实相对各向同性的F-肌动蛋白网络足够强大以稳定细胞。
