Small J V, Herzog M, Anderson K
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
J Cell Biol. 1995 Jun;129(5):1275-86. doi: 10.1083/jcb.129.5.1275.
From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.
从近期对游动的鱼类角膜细胞的研究中提出，肌动蛋白丝的动态周转是通过成核-释放机制进行的，这预测在整个片状伪足中存在短（小于0.5微米）的丝（泰里奥特，J. A.，和T. J. 米奇森。1991年。《自然》（伦敦）。352:126 - 131）。我们通过在电子显微镜下研究整装角膜细胞细胞骨架的结构以及在光镜下研究经各种固定处理后用鬼笔环肽标记的细胞，对该模型进行了测试。经Triton提取产生的负染角膜细胞细胞骨架的显微照片显示，片状伪足中的肌动蛋白丝初步组织成二维正交网络，这些丝与细胞前沿呈约45度角。通过添加外源肌动蛋白在角膜细胞细胞骨架前沿生长的肌动蛋白丝条纹在用肌球蛋白亚片段-1标记时显示出均匀的极性，这与肌动蛋白丝的快速生长端邻接前缘一致。从片状伪足的中部区域到核周区域观察到丝密度稳步下降，并且在丝密度较低的更后部区域的图像中，可以看到许多肌动蛋白丝长度至少为几微米。对整个片状伪足上荧光鬼笔环肽染色强度分布的定量分析表明，丝密度梯度以及F-肌动蛋白的绝对含量取决于固定方法。在先用固定剂固定然后用Triton提取的细胞中，从片状伪足的前端到后端观察到鬼笔环肽染色的陡峭梯度。按照获得电子显微镜图像所需的方案，即先用Triton提取然后固定，片状伪足前部的鬼笔环肽染色显著且优先减少。这导致丝密度梯度降低，与在电子显微镜中看到的一致，并表明在Triton提取过程中约45%的丝状肌动蛋白丢失。我们得出结论，首先，丝的组织和长度分布不支持成核释放模型，而更符合以分级长度的肌动蛋白丝为特征的踏车运动型机制。其次，我们认为两层丝构成片状伪足；一层较低的、与腹侧膜相关的稳定层和一层与背侧膜相关的上层，上层由长度范围比下层短的丝组成，并且主要在Triton处理中丢失。
