Abstract
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.
Highlights
In order to guide the outgrowth of neuronal axons to their correct destinations, their distal ends form highly motile extensions—the growth cones, which are guided by soluble or non-soluble repellent or attractant cues [1,2]
Growth cones of chicken dorsal root ganglia (DRG) were analyzed after 3 days of cultivation in nutrient medium supplemented with growth factors
We can conclude that vascular endothelial growth factor (VEGF) stimulates cofilin within the growth cone, and that growth cones with constitutively inactive cofilin do not grow upon VEGF stimulation
Summary
In order to guide the outgrowth of neuronal axons to their correct destinations, their distal ends form highly motile extensions—the growth cones, which are guided by soluble or non-soluble repellent or attractant cues [1,2]. Activated growth cones explore the immediate environment with numerous filopodia, thin finger-like extensions of the plasma membrane, which are stabilized by bundles of filamentous actin (F-actin). Actin polymerization is a directional process powered by the hydrolysis of ATP (adenosine triphosphate) by the actin subunits immediately after their addition to the so-called fast growing or plus end, whereas the dissociation of actin subunits from the filament occurs from the minus end. This process is termed actin treadmilling or cycling, and can provide protrusive forces when the plus ends are oriented towards the plasma membrane [3]. Polymerization of monomeric actin is promoted by nuclei of small actin oligomers, whose formation and stabilization are favored by nucleating proteins, such as the formins and the Arp2/3 complex, which is composed of seven different protein subunits [4,5,6]
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