Forscher P, Kaczmarek L K, Buchanan J A, Smith S J
Howard Hughes Medical Institute Section of Molecular Neurobiology, Yale School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1987 Nov;7(11):3600-11. doi: 10.1523/JNEUROSCI.07-11-03600.1987.
This report examines cAMP-induced regulation of directed organelle transport in bag cell neuron growth cones using video-enhanced differential interference contrast (DIC) microscopy (Allen et al., 1981; Inoue, 1981) and digital image analysis techniques. Under control conditions, organelle transport is evident in the central cytoplasmic regions of bag cell neuron growth cones, but not in lamellae. Motility of lamellae takes the form of slow (less than 0.01 micron/sec) extension of margins and ruffling motions that propagate as waves (velocity, approximately 0.07 micron/sec) in a retrograde direction. Application of forskolin and a phosphodiesterase (PDE) inhibitor at concentrations known to induce changes in bag cell protein phosphorylation resulted in (1) rapid extension of directed organelle transport into lamellae, and (2) inhibition of the retrograde ruffling waves. These changes effected transformation of lamellae into neurite endings packed with microtubules and organelles, a large proportion of which appeared to be neurosecretory granules. The effects were reversible, dose-dependent, potentiated by a variety of PDE inhibitors, and mimicked by 6-N-butyl-8-benzyl-thio-cAMP (BT-cAMP). Though forskolin may normally promote depolarization and Ca entry, these changes in growth cone structure are not secondary to influx of external Ca, as they persist in Ca-free/EGTA solutions; furthermore, they do not resemble the effects of depolarization induced by perfusion with elevated K solutions. The cAMP-induced changes in growth cone morphology that we report here suggest a possible role for protein phosphorylation in promoting growth cone differentiation and structural changes accompanying secretion.
本报告利用视频增强微分干涉相差显微镜（DIC显微镜）（Allen等人，1981年；Inoue，1981年）和数字图像分析技术，研究了环磷酸腺苷（cAMP）诱导的袋状细胞神经元生长锥中定向细胞器运输的调控。在对照条件下，细胞器运输在袋状细胞神经元生长锥的中央细胞质区域明显可见，但在片状伪足中则不然。片状伪足的运动表现为边缘缓慢（小于0.01微米/秒）伸展以及以波的形式逆行传播的褶皱运动（速度约为0.07微米/秒）。应用已知能诱导袋状细胞蛋白质磷酸化变化的浓度的福斯可林和磷酸二酯酶（PDE）抑制剂，导致（1）定向细胞器运输迅速扩展到片状伪足中，以及（2）逆行褶皱波受到抑制。这些变化使片状伪足转变为充满微管和细胞器的神经突末梢，其中很大一部分似乎是神经分泌颗粒。这些效应是可逆的、剂量依赖性的，可被多种PDE抑制剂增强，并可被6 - N - 丁基 - 8 - 苄基 - 硫代 - cAMP（BT - cAMP）模拟。尽管福斯可林通常可能促进去极化和钙内流，但生长锥结构的这些变化并非外部钙流入的继发结果，因为它们在无钙/乙二醇双乙酸盐（EGTA）溶液中仍然持续存在；此外，它们与用高钾溶液灌注诱导的去极化效应不同。我们在此报告的cAMP诱导的生长锥形态变化表明，蛋白质磷酸化在促进生长锥分化和伴随分泌的结构变化中可能发挥作用。
