Lewis A K, Bridgman P C
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Cell Biol. 1992 Dec;119(5):1219-43. doi: 10.1083/jcb.119.5.1219.
The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.
利用负染和冷冻蚀刻电子显微镜技术，通过一种通透化方案研究了神经元生长锥中肌动蛋白丝的组织和极性。当用视频增强微分干涉相差显微镜观察培养的神经生长锥时，该方案几乎不会引起形态上可检测到的变化。片足肌动蛋白细胞骨架由两个不同的亚群组成：一群宽度为40 - 100纳米的丝状束从前沿放射状伸出，另一群分支短丝填充在背侧和腹侧膜表面之间的空间。这两个亚群共同形成了在扩展的片足内所见的三维结构网络。肌动蛋白丝与腹侧膜表面的相互作用通过膜相关蛋白沿着丝的长度发生。长的成束丝状群体主要参与这些相互作用。任一群体的丝末端似乎仅在前沿与膜相互作用；这种相互作用由球状的不溶于 Triton 的物质介导。通过用肌球蛋白 S1 或重酶解肌球蛋白进行标记来确定肌动蛋白丝的极性。先前的报道表明，运动细胞中肌动蛋白丝的极性是一致的，带刺末端朝向前沿。我们观察到生长锥片足内的肌动蛋白丝极性并不一致；尽管主要取向是带刺末端朝向前沿（47 - 56%），但22 - 25%的丝具有相反的取向，其尖末端朝向前沿，19 - 31%与前沿平行。这两个肌动蛋白丝群体显示出不同的极性分布：较长的丝似乎主要以其带刺末端朝向前沿取向，而短丝似乎是随机取向的。两个丝状群体不同的长度、组织和极性表明它们在稳定性和功能上存在差异。成束的长丝群体似乎更多地位于腹侧并与膜蛋白接触，可能比较短的分支丝群体更稳定。长的成束丝的位置、组织和极性表明它们可能对于片足的扩展以及由受体与底物粘附分子介导的张力产生是必需的。
