Cramer L P, Siebert M, Mitchison T J
The Randall Institute, Kings College London, University of London, United Kingdom.
J Cell Biol. 1997 Mar 24;136(6):1287-305. doi: 10.1083/jcb.136.6.1287.
We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25-2.5 microm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 microm, but can reach up to about 30 microm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward. By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body. This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.
我们通过S1标记、连续切片电子显微镜以及荧光光活化技术，确定了原代运动性心脏成纤维细胞中肌动蛋白丝的结构组织和动态行为。正如预期的那样，这些细胞片状伪足中的肌动蛋白丝具有统一的极性，其带刺端朝前。在片状伪足、细胞体和尾部，存在两种可观察到的肌动蛋白丝组织类型。一种数量较少的类型位于质膜内表面，由短的、重叠的肌动蛋白束（0.25 - 2.5微米）组成，其极性从统一的带刺端朝前反复交替为统一的尖端朝前。这种组织类型类似于我们在非运动细胞（PtK2细胞）中展示的肌动蛋白丝束（应力纤维）的组织类型，也类似于已知的肌肉肌节组织。运动性心脏成纤维细胞中数量较多的肌动蛋白丝组织类型大多位于腹侧，由长的、重叠的束（平均13微米，但可达约30微米）组成，这些束跨越细胞的长度。这种数量较多的类型具有一种新的渐变极性组织。在每个肌动蛋白束中，极性沿着束的长度逐渐变化。束中任何给定位置的实际肌动蛋白丝极性由其在细胞中的位置决定；越靠近细胞前端，肌动蛋白丝的带刺端朝前的越多。通过对运动性心脏成纤维细胞进行光活化标记，正如在片状伪足中预期的那样，肌动蛋白丝相对于底物向后流动。在片状伪足中，随着成纤维细胞运动，所有标记并观察到的肌动蛋白丝相对于底物保持静止。在运动性成纤维细胞的细胞体中有两种动态的肌动蛋白丝群体：一种保持静止，另一种相对于底物以细胞体的速度向前移动。这是首次在整个运动细胞中确定肌动蛋白丝的结构组织和动态。渐变极性肌动蛋白丝束的组织、动态和相对丰度对原代心脏成纤维细胞运动过程中运动力的产生具有重要意义。
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