Cortese J D, Frieden C
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Cell Motil Cytoskeleton. 1990;17(3):236-49. doi: 10.1002/cm.970170310.
We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.
我们之前已经证实[科尔特斯和弗里登，《细胞生物学杂志》107:1477 - 1487，1988]，在剪切力作用下形成的肌动蛋白凝胶是微不均匀的。在本研究中，使用荧光光漂白恢复和视频显微镜研究了交联（通过鸡胗细丝蛋白）、切断（通过血浆凝溶胶蛋白）以及剪切对肌动蛋白微不均匀性的影响。我们发现细丝蛋白和剪切力通过不同机制形成微不均匀的F - 肌动蛋白：凝溶胶蛋白凝胶。细丝蛋白对肌动蛋白：凝溶胶蛋白丝的成束作用可以通过丝间结合导致细丝表观长度增加来解释，这导致细丝组织成各向异性相时聚合物数浓度降低。细丝蛋白与肌动蛋白丝之间可能也存在一些丝内结合，并且在相同聚合条件下，那些被细丝蛋白包被的丝比肌动蛋白固定得更慢。F - 肌动蛋白/凝溶胶蛋白丝的长度似乎是控制相对于网络形成的成束程度的一个主要因素。相比之下，剪切力对肌动蛋白：凝溶胶蛋白丝微不均匀性的影响与我们之前的观点一致，即剪切力使肌动蛋白丝排列整齐，从而允许丝 - 丝相互作用和相形成发生。短丝无法组织成分支状肌动蛋白网络，但在低剪切力下它们可以形成大聚集体。较长的肌动蛋白丝将以具有不同分支水平的网络形式存在，并且对剪切力不太敏感。剪切力场强度对肌动蛋白空间分布的影响可能涉及肌动蛋白分子和束的取向逐渐变得更加随机。在低剪切速率（0.04 - 1.39 s⁻¹）下，样品中会形成规则图案，而在较高剪切速率（大于10 s⁻¹）下会变得非常不规则。我们在此提出，肌动蛋白结合蛋白和剪切力可以通过它们对表观长度和局部丝浓度的影响来控制各向同性网络和各向异性相之间的转变，并且这种转变可能对细胞对机械应力的抗性产生重大影响。
