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Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Lalli G, Hall A - J. Cell Biol. (2005)

Bottom Line: Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching.In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons.Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, England, UK.

ABSTRACT
Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

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GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).
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fig7: GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).

Mentions: To further test this conclusion, constitutively active Ral and a cGAP-43 mutant that cannot be phosphorylated on Ser42 (S42A; Widmer and Caroni, 1993) were coinjected in SCG neurons plated on laminin. The striking increase in branching caused by active Ral was blocked (Fig. 6, A and B). Finally, a phosphomimetic mutant of cGAP-43 (S42D; Widmer and Caroni, 1993) was able to fully rescue the decreased branching caused by inhibition of RalB in cells plated on laminin (Fig. 7, A and B). Interestingly, rescue of inhibition of the RalA isoform was only partial (Fig. 7, A and B). In contrast, wild-type cGAP-43 did not rescue the effects of dominant-negative Ral (unpublished data). Similar results were obtained for neurons initially plated on polyornithine, microinjected, and left to express for a few hours before laminin addition (Fig. 7, C and D). Together, these results place GAP-43 downstream of an integrin- and Ral-dependent pathway leading to neurite branching.


Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Lalli G, Hall A - J. Cell Biol. (2005)

GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).
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fig7: GAP-43 acts downstream of Ral. (A) A phosphomimetic GAP-43 protein, cGAP-43(S42D), restores branching in neurons plated on laminin and expressing dominant-negative Ral. Shown here are neurons expressing EGFP-F (top left), RalA28N (top middle), or RalB28N (bottom left) and RalA28N or RalB28N and cGAP-43(S42D) (right). Cells are stained with an anti-myc antibody to detect mutant Ral and an anti–cGAP-43 antibody. Bar, 50 μm. (B) Quantitative analysis of branching in cells plated on laminin and injected with single Ral isoforms or with Ral and cGAP-43(S42D) (means ± SEM: GFP, 3.47 ± 0.37; RalA28N, 1.74 ± 0.52; RalA28N + cGAP-43(S42D), 2.42 ± 0.31; RalB28N, 1.56 ± 0.41; RalB28N + cGAP-43(S42D), 3.72 ± 0.35; *, P < 0.04; **, P < 0.001). (C) SCG neurons were plated on polyornithine, microinjected, and left to express the indicated proteins for 5 h before laminin addition. Expression of dominant-negative Ral decreases branching compared with EGFP-F–expressing cells (top, compare middle and right with left). Branching is restored in cells coexpressing cGAP-43(S42D) (middle and bottom rows). Bar, 100 μm. (D) Quantitative analysis of branching in neurons initially plated on polyornithine and injected with single Ral isoforms or with Ral and cGAP-43(S42A) before laminin addition (means ± SEM: GFP, 2.64 ± 0.35; RalA28N, 1.05 ± 0.32; RalA28N + cGAP-43(S42D), 1.82 ± 0.40; RalB28N, 1.74 ± 0.30; RalB28N + cGAP-43(S42D), 2.73 ± 0.45; **, P < 0.01).
Mentions: To further test this conclusion, constitutively active Ral and a cGAP-43 mutant that cannot be phosphorylated on Ser42 (S42A; Widmer and Caroni, 1993) were coinjected in SCG neurons plated on laminin. The striking increase in branching caused by active Ral was blocked (Fig. 6, A and B). Finally, a phosphomimetic mutant of cGAP-43 (S42D; Widmer and Caroni, 1993) was able to fully rescue the decreased branching caused by inhibition of RalB in cells plated on laminin (Fig. 7, A and B). Interestingly, rescue of inhibition of the RalA isoform was only partial (Fig. 7, A and B). In contrast, wild-type cGAP-43 did not rescue the effects of dominant-negative Ral (unpublished data). Similar results were obtained for neurons initially plated on polyornithine, microinjected, and left to express for a few hours before laminin addition (Fig. 7, C and D). Together, these results place GAP-43 downstream of an integrin- and Ral-dependent pathway leading to neurite branching.

Bottom Line: Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching.In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons.Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, England, UK.

ABSTRACT
Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

Show MeSH
Related in: MedlinePlus