Lambrechts Anja, Jonckheere Veronique, Peleman Christa, Polet Debby, De Vos Winnok, Vandekerckhove Joël, Ampe Christophe
Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Belgium.
J Cell Sci. 2006 Apr 15;119(Pt 8):1570-8. doi: 10.1242/jcs.02884. Epub 2006 Mar 28.
Differentiating neurons extend membrane protrusions that develop into growing neurites. The driving force for neurite outgrowth is the dynamic actin cytoskeleton, which is regulated by actin-binding proteins. In this study, we describe for the first time, the role of profilin I and its ligand interactions in neuritogenesis of PC12 cells. High-level overexpression of wild-type profilin I had an inhibitory effect on neurite outgrowth. Low levels of profilin I did not disturb this process, but these cells developed many more filopodia along the neurite shafts. Low-level overexpression of mutant forms of profilin I changed one or more aspects of PC12 differentiation. Expression of a profilin I mutant that is defective in actin binding (profilin I(R74E)) decreased neurite length and strongly inhibited filopodia formation. Cells expressing mutants defective in binding proline-rich ligands (profilin I(W3A) and profilin I(R136D)) differentiated faster, developed more and longer neurites and more branches. The profilin I(R136D) mutant, which is also defective in phosphatidylinositol 4,5-bisphosphate binding, enhanced neurite outgrowth even in the absence of NGF. Parental PC12 cells treated with the ROCK inhibitor Y27632, differentiate faster and display longer neurites and more branches. Similar effects were seen in cells expressing profilin I(WT), profilin I(W3A) and profilin I(R74E). By contrast, the profilin I(R136D)-expressing cells were insensitive to the ROCK inhibitor, suggesting that regulation of profilin I by phosphatidylinositol 4,5-bisphosphate metabolism is crucial for proper neurite outgrowth. Taken together, our data show the importance of the interaction of profilin I with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate in neuronal differentiation of PC12 cells.
正在分化的神经元会延伸出膜突起，这些突起会发育成生长中的神经突。神经突生长的驱动力是动态的肌动蛋白细胞骨架，它受肌动蛋白结合蛋白调控。在本研究中，我们首次描述了肌动蛋白 Profilin I 及其配体相互作用在 PC12 细胞神经突形成中的作用。野生型 Profilin I 的高水平过表达对神经突生长有抑制作用。低水平的 Profilin I 不会干扰这一过程，但这些细胞在神经突轴上会形成更多的丝状伪足。Profilin I 突变体的低水平过表达改变了 PC12 分化的一个或多个方面。一种在肌动蛋白结合方面有缺陷的 Profilin I 突变体（Profilin I(R74E)）的表达会缩短神经突长度并强烈抑制丝状伪足的形成。表达在结合富含脯氨酸的配体方面有缺陷的突变体（Profilin I(W3A) 和 Profilin I(R136D)）的细胞分化更快，形成更多更长的神经突和更多分支。Profilin I(R136D) 突变体在磷脂酰肌醇 4,5 - 二磷酸结合方面也有缺陷，即使在没有神经生长因子（NGF）的情况下也能增强神经突生长。用 ROCK 抑制剂 Y27632 处理的亲代 PC12 细胞分化更快，显示出更长的神经突和更多分支。在表达 Profilin I(WT)、Profilin I(W3A) 和 Profilin I(R74E) 的细胞中也观察到了类似的效果。相比之下，表达 Profilin I(R136D) 的细胞对 ROCK 抑制剂不敏感，这表明磷脂酰肌醇 4,5 - 二磷酸代谢对 Profilin I 的调节对于正常的神经突生长至关重要。综上所述，我们的数据表明 Profilin I 与肌动蛋白、富含脯氨酸的蛋白质以及磷脂酰肌醇 4,5 - 二磷酸的相互作用在 PC12 细胞的神经元分化中具有重要意义。
