生长锥是延伸轴突的高度运动尖端,对于神经网络的形成至关重要。三维结构照明显微镜,一种超分辨率光学显微镜,具有克服光学衍射限制的分辨率(ca。200nm)的常规光学显微镜,非常适合研究细胞内事件的分子动力学。使用这种技术,我们在生长锥内发现了一种沿z轴分布的新型丝足病(“z-丝足病”)。Z-丝状伪足通常朝向轴突生长的方向,没有连接到底层,自发突出,没有微管侵入,寿命比传统的丝足短得多。Z-丝状伪足的形成和动力学受肌动蛋白调节蛋白,如血管扩张剂刺激的磷蛋白,Fascin,还有cofilin.发色团辅助的激光灭活cofilin诱导了z-filopodia的快速周转。轴突导向受体,Neuropilin-1,集中在z-丝状足中,并与它们一起运输,而它的配体,信号蛋白-3A,被选择性地束缚在他们身上。与z-filopodia相关的膜结构域也是特化的,类似于脂筏,它们的行为与神经纤毛蛋白1的行为密切相关。结果表明,z-filopodia具有独特的周转特性,和xy-filopodia不同,不作为轴突延伸的力生成结构。
A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis (\"z-filopodia\") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.