ACTIN FILAMENT ORGANIZATION IN THE FISH KERATOCYTE LAMELLIPODIUM
ACTIN FILAMENT ORGANIZATION IN THE FISH KERATOCYTE LAMELLIPODIUM
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DOI：
10.1083/jcb.129.5.1275
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发表时间：
1995-06-01
影响因子：
7.8
通讯作者：
ANDERSON, K
中科院分区：
文献类型：
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作者：
SMALL, JV;HERZOG, M;ANDERSON, K
从最近对迁移的鱼角膜细胞的研究中提出，肌动蛋白丝的动态更新是通过成核释放机制发生的，该机制预测在整个板状伪足中存在短（小于0.5 μ m）丝（Theriot，J.A.，和T. J·米奇森1991.自然（借贷），352：126-131），我们通过在电子显微镜下研究整个角膜细胞骨架的结构和在光学显微镜下研究各种固定后鬼笔环肽标记的细胞来测试该模型。通过Triton提取产生的负染色角膜细胞骨架的显微照片显示，板状伪足的肌动蛋白丝被组织成二维正交网络的第一近似值，其中所述丝与细胞正面成约45度角。肌动蛋白丝的边缘生长到角膜基质细胞骨架的前边缘，通过添加外源性肌动蛋白显示出均匀的极性时，装饰肌球蛋白亚片段-1，与邻接的前边缘的肌动蛋白丝的快速生长的末端一致。观察到纤维密度从中间开始稳定下降，在核周区和较低纤维密度的较后区的图像中，可以看到许多肌动蛋白纤维的长度至少为几微米。荧光鬼笔环肽染色强度分布的定量分析表明，纤维密度的梯度以及纤维的绝对含量F-肌动蛋白依赖于固定方法。在首先固定然后用Triton提取的细胞中，从板状伪足的前部到后部观察到陡峭的鬼笔环肽染色梯度。使用获得电子显微镜图像所需的方案，即Triton提取后固定，鬼笔环肽染色在板状伪足的前部显著且优先减少。这导致在较低的梯度丝密度，在电子显微镜中看到的一致，并表示约45%的损失的丝状肌动蛋白在Triton extraction.We的结论，第一丝的组织和长度分布不支持成核释放模型，但更符合一个milling型的运动机制，具有肌动蛋白丝的梯度长度。第二，我们建议，两层的细丝组成的板状伪足;一个较低的，稳定的层与腹侧膜和上层与背侧膜，是由细丝的长度范围较短的比下层，这主要是失去了海卫一。
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 mu m) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lend.), 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.