Kuhn T B, Williams C V, Dou P, Kater S B
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
J Neurosci. 1998 Jan 1;18(1):184-94. doi: 10.1523/JNEUROSCI.18-01-00184.1998.
During development, growth cones navigate to their targets via numerous interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisions are poorly understood. We have examined the role of intracellular Ca2+ in laminin (LN)-mediated growth cone navigation in vitro, using chick dorsal root ganglion neurons. Subsequent to contacting LN-coated beads with filopodia, growth cones displayed a series of stereotypic changes in behavior, including turning toward LN-coated beads and a phase of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turning required an influx of extracellular Ca2+, likely in filopodia with LN contact, and activation of calmodulin (CaM). Surprisingly, fluorescent Ca2+ imaging revealed no LN-induced rise in intracellular Ca2+ in filopodia attached to their parent growth cone. However, isolation of filopodia by laser-assisted transection unmasked a rapid, LN-specific rise in intracellular Ca2+ (+73 +/- 11 nM). Additionally, a second, sustained rise in intracellular Ca2+ (+62 +/- 8 nM) occurred in growth cones, with a distinct delay 28 +/- 3 min after growth cone filopodia contacted LN-coated beads. This delayed, sustained Ca2+ signal paralleled the phase of increased rates of outgrowth, and both events were sensitive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) with 2 microM KN-62. We propose that LN-mediated growth cone guidance can be attributed, in part, to two temporally and functionally distinct Ca2+ signals linked by a signaling cascade composed of CaM and CaM-kinase II.
在发育过程中，生长锥通过与分子引导线索的大量相互作用导航至其目标，但生长锥如何将引导信息转化为导航决策的机制仍知之甚少。我们利用鸡背根神经节神经元，在体外研究了细胞内Ca2+在层粘连蛋白（LN）介导的生长锥导航中的作用。丝状伪足与包被LN的珠子接触后，生长锥表现出一系列刻板的行为变化，包括转向包被LN的珠子以及在包被LN的珠子处暂停后生长速率增加的阶段。药理学方法表明，LN介导的生长锥转向需要细胞外Ca2+的流入，可能是在与LN接触的丝状伪足中，以及钙调蛋白（CaM）的激活。令人惊讶的是，荧光Ca2+成像显示，附着于其母生长锥的丝状伪足中没有LN诱导的细胞内Ca2+升高。然而，通过激光辅助横切分离丝状伪足揭示了细胞内Ca2+的快速、LN特异性升高（+73±11 nM）。此外，生长锥中发生了第二次持续细胞内Ca2+升高（+62±8 nM），在生长锥丝状伪足接触包被LN的珠子后28±3分钟有明显延迟。这种延迟的、持续的Ca2+信号与生长速率增加的阶段平行，并且这两个事件对用2 μM KN-62抑制Ca2+/CaM依赖性蛋白激酶II（CaM激酶II）敏感。我们提出，LN介导的生长锥引导部分可归因于由CaM和CaM激酶II组成的信号级联连接的两个时间和功能上不同的Ca2+信号。
