Welnhofer E A, Zhao L, Cohan C S
Department of Anatomy and Cell Biology, University at Buffalo, State University of New York, Buffalo, New York 14214, USA.
J Neurosci. 1999 Sep 15;19(18):7971-82. doi: 10.1523/JNEUROSCI.19-18-07971.1999.
The ability of calcium (Ca(2+)) to effect changes in growth cone motility requires remodeling of the actin cytoskeleton. To understand the mechanisms involved, we evaluated the effect of elevated intracellular calcium (Ca(2+)) on actin bundle dynamics, organization, and retrograde flow in the large growth cones of identified Helisoma neurons. Depolarization with 15 mM KCl (high K(+)) for 30 min caused a rapid and sustained increase in Ca(2+) and resulted in longer filopodia, shorter actin ribs, and a decrease in lamellipodia width. Time-lapse microscopy revealed that increasing Ca(2+) affected actin bundle dynamics differently at the proximal and distal ends. Filopodial lengthening resulted from assembly-driven elongation of actin bundles whereas actin rib shortening resulted from a distal shift in the location of breakage. Buckling of ribs occurred before breakage, suggesting nonuniform forces were applied to ribs before shortening. Calcium (Ca(2+)) influx also resulted in a decrease in density of F-actin in bundles, as determined by contrast changes in ribs imaged by differential interference contrast microscopy and fluorescent intensity changes in rhodamine-labeled ribs. The velocity of retrograde flow decreased by 50% after elevation of Ca(2+). However, no significant change in retrograde flow occurred when the majority of changes in actin bundles were blocked by phalloidin. This suggests that inhibition of retrograde flow resulted from Ca(2+)-induced changes in the actin cytoskeleton. These results implicate Ca(2+) as a regulator of actin dynamics and, as such, provide a mechanism by which Ca(2+) can influence growth cone motility and behavior.
钙（Ca(2+)）影响生长锥运动性变化的能力需要肌动蛋白细胞骨架的重塑。为了理解其中涉及的机制，我们评估了细胞内钙浓度升高（Ca(2+)）对已鉴定的椎实螺神经元大型生长锥中肌动蛋白束动力学、组织和逆行流动的影响。用15 mM KCl（高钾）去极化30分钟导致Ca(2+)迅速且持续升高，并导致丝状伪足变长、肌动蛋白肋变短以及片状伪足宽度减小。延时显微镜显示，Ca(2+)升高对近端和远端的肌动蛋白束动力学影响不同。丝状伪足的延长是由肌动蛋白束的组装驱动伸长导致的，而肌动蛋白肋的缩短是由断裂位置的远端移位导致的。肋在断裂前发生弯曲，表明在缩短之前对肋施加了不均匀的力。通过微分干涉对比显微镜成像的肋的对比度变化以及罗丹明标记的肋的荧光强度变化确定，钙（Ca(2+)）内流还导致束中F-肌动蛋白密度降低。Ca(2+)升高后，逆行流动速度降低了50%。然而，当肌动蛋白束的大部分变化被鬼笔环肽阻断时，逆行流动没有显著变化。这表明逆行流动的抑制是由Ca(2+)诱导的肌动蛋白细胞骨架变化引起的。这些结果表明Ca(2+)是肌动蛋白动力学的调节因子，因此提供了一种Ca(2+)可以影响生长锥运动性和行为的机制。
