Institute of Anatomy, Department of Cytology, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany.
Research Group Molecular Cardiology, University Hospital Bergmannsheil and St. Josef Hospital, c/o Clinical Pharmacology, Ruhr-University, 44780 Bochum, Germany.
Int J Mol Sci. 2018 Jan 27;19(2):384. doi: 10.3390/ijms19020384.
A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott-Aldrich-Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.
轴突生长锥是神经元网络发育和再生的关键神经元结构。它受不同导向线索的影响，朝着预定的方向生长，以到达最终目的地。其中一种线索是血管内皮生长因子（VEGF），它被确定为生长锥运动的正向效应物。这些正向效应主要通过肌动蛋白网络的重组来介导。本研究表明，VEGF 可引发鸡背根神经节（DRG）中轴突生长锥内的 cofilin 和 Arp2/3 复合物与肌动蛋白细胞骨架的紧密共定位。GFP（绿色荧光蛋白）-cofilin 和 RFP（红色荧光蛋白）-LifeAct 的活体细胞成像显示，这两种标记蛋白在生长锥内迅速重新分布，并在 VEGF 补充后显示出一致的分布模式。通过阻断 LIM 激酶（LIMK）活性阻断 cofilin 信号的上游，导致生长锥显示出退行性生长行为。GFP-p16b（Arp2/3 复合物的 a 亚基）和 RFP-LifeAct 的微注射显示，这两种蛋白在 VEGF 刺激后几分钟内重新分布到生长锥的片状伪足中。在 VEGF 存在的情况下，通过抑制 N-WASP（神经元 Wiskott-Aldrich-Scott 蛋白）抑制 Arp2/3 复合物到信号，导致生长锥回缩。因此，cofilin 和 Arp2/3 复合物似乎是 VEGF 信号到 DRG 生长锥肌动蛋白细胞骨架的下游效应蛋白。我们的数据表明，VEGF 同时影响到肌动蛋白细胞骨架信号的不同途径，因为 cofilin 的激活是通过 LIMK 的抑制来实现的，而 Arp2/3 的激活是通过 N-WASP 的刺激来实现的。
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