Tornieri Karine, Welshhans Kristy, Geddis Matthew S, Rehder Vincent
Department of Biology, Georgia State University, Atlanta, Georgia 30302, USA.
Cell Motil Cytoskeleton. 2006 Apr;63(4):173-92. doi: 10.1002/cm.20115.
Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.
据报道，磷脂酰肌醇-3-激酶（PI-3K）在体内和体外均会影响神经突生长。在此，我们研究了PI-3K在体外影响特定蜗牛神经元神经突生长和生长锥运动的信号通路。用渥曼青霉素（2 microM）或LY 294002（25 microM）抑制PI-3K会导致丝状伪足显著伸长以及神经突生长减缓。分别使用干扰肌动蛋白聚合的细胞松弛素和干扰肌球蛋白II活性的blebbistatin进行的实验表明，PI-3K抑制导致的丝状伪足伸长依赖于肌动蛋白聚合。抑制位于PI-3K下游的关键激酶，如Akt、ROCK和MEK，也会导致丝状伪足显著伸长和神经突生长减缓。另一个生长锥参数，丝状伪足数量，不受PI-3K、Akt、ROCK或MEK抑制的影响。对生长锥行为的详细研究表明，抑制PI-3K、Akt、ROCK和MEK诱导的丝状伪足伸长是通过增加两个运动参数实现的：丝状伪足延伸的速率（延伸速率）和丝状伪足伸长所花费的时间。虽然抑制ROCK或Akt（两者均由PI-3K的脂质激酶活性激活）和MEK（由PI-3K的蛋白激酶活性激活）具有累加效应，但同时抑制Akt和ROCK则没有累加效应。我们进一步证明，所研究的对丝状伪足动力学的影响与钙无关。综上所述，我们的结果表明，抑制PI-3K信号会导致丝状伪足伸长和神经突推进减缓，这让人联想到生长锥的探索行为。
