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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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Ral is activated by laminin and is required for neurite branching promoted by laminin. (A) Phase-contrast pictures of SCG neurons taken 3 h after plating on polyornithine (polorn), polyornithine and laminin (lam), or polyornithine and collagen (coll). Note the visible neurite outgrowth and branching of the laminin-plated neurons compared with cells growing on the other substrates. Bar, 20 μm. (B) SCG neurons were lysed 3 h after plating on the indicated substrates. The amount of RalA-GTP in the lysates was determined by affinity purification with the Ral binding domain of RalBP1 and immunoblotting with anti-RalA antibodies. A representative blot is shown (left), together with the quantification of data of three independent experiments (right; means ± SEM; *, P < 0.005). (C, top) SCG neurons stained for tubulin and photographed 24 h after plating on polyornithine or polyornithine and laminin. Cells plated on polyornithine show limited neurite outgrowth and branching compared with neurons growing on laminin. Bar, 100 μm. (bottom) SCG neurons plated on polyornithine and expressing Rlf-CAAX display extensive branching (middle), an effect absent in neurons expressing EGFP-F (left) or inactive Rlf (Rlf-ΔCAT-CAAX; right). Bar, 50 μm. (D) SCG neurons plated on polyornithine were left to express the indicated proteins for 5 h before laminin addition. Pictures were taken after overnight expression. Neurite branching triggered by laminin as observed in cells expressing EGFP-F (top left) is impaired in neurons expressing dominant-negative Ral isoforms (top right and bottom left). (bottom right) A quantitative analysis of neurite branching in five independent laminin-addition experiments (means ± SEM: GFP, 2.19 ± 0.18; RalA28N, 0.84 ± 0.09; RalB28N, 0.95 ± 0.10; **, P < 0.0001). Bar, 30 μm.
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fig3: Ral is activated by laminin and is required for neurite branching promoted by laminin. (A) Phase-contrast pictures of SCG neurons taken 3 h after plating on polyornithine (polorn), polyornithine and laminin (lam), or polyornithine and collagen (coll). Note the visible neurite outgrowth and branching of the laminin-plated neurons compared with cells growing on the other substrates. Bar, 20 μm. (B) SCG neurons were lysed 3 h after plating on the indicated substrates. The amount of RalA-GTP in the lysates was determined by affinity purification with the Ral binding domain of RalBP1 and immunoblotting with anti-RalA antibodies. A representative blot is shown (left), together with the quantification of data of three independent experiments (right; means ± SEM; *, P < 0.005). (C, top) SCG neurons stained for tubulin and photographed 24 h after plating on polyornithine or polyornithine and laminin. Cells plated on polyornithine show limited neurite outgrowth and branching compared with neurons growing on laminin. Bar, 100 μm. (bottom) SCG neurons plated on polyornithine and expressing Rlf-CAAX display extensive branching (middle), an effect absent in neurons expressing EGFP-F (left) or inactive Rlf (Rlf-ΔCAT-CAAX; right). Bar, 50 μm. (D) SCG neurons plated on polyornithine were left to express the indicated proteins for 5 h before laminin addition. Pictures were taken after overnight expression. Neurite branching triggered by laminin as observed in cells expressing EGFP-F (top left) is impaired in neurons expressing dominant-negative Ral isoforms (top right and bottom left). (bottom right) A quantitative analysis of neurite branching in five independent laminin-addition experiments (means ± SEM: GFP, 2.19 ± 0.18; RalA28N, 0.84 ± 0.09; RalB28N, 0.95 ± 0.10; **, P < 0.0001). Bar, 30 μm.

Mentions: Because integrin activation has been implicated in the regulation of neurite outgrowth and branching (Ivins et al., 2000), we tested the influence of different substrates on the levels of Ral activation. SCG neurons were plated on plastic dishes coated with polyornithine only or polyornithine plus either laminin or collagen. 3 h after plating, neuronal morphology varied according to the substrate (Fig. 3 A). SCGs plated on collagen or on polyornithine appeared mainly round with very short processes (Fig. 3 A, top left and bottom). In contrast, cells growing on laminin had longer, branched neurites (Fig. 3 A, top right). To quantify the levels of active Ral under these different conditions, we used a fragment of RalBP1 known to preferentially bind to Ral-GTP as an affinity reagent (Tian et al., 2002). Ral-GTP levels in neurons plated on laminin were approximately fourfold higher than on the other substrates, whereas the total amount of Ral in lysates from different samples was comparable (Fig. 3 B).


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

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

Ral is activated by laminin and is required for neurite branching promoted by laminin. (A) Phase-contrast pictures of SCG neurons taken 3 h after plating on polyornithine (polorn), polyornithine and laminin (lam), or polyornithine and collagen (coll). Note the visible neurite outgrowth and branching of the laminin-plated neurons compared with cells growing on the other substrates. Bar, 20 μm. (B) SCG neurons were lysed 3 h after plating on the indicated substrates. The amount of RalA-GTP in the lysates was determined by affinity purification with the Ral binding domain of RalBP1 and immunoblotting with anti-RalA antibodies. A representative blot is shown (left), together with the quantification of data of three independent experiments (right; means ± SEM; *, P < 0.005). (C, top) SCG neurons stained for tubulin and photographed 24 h after plating on polyornithine or polyornithine and laminin. Cells plated on polyornithine show limited neurite outgrowth and branching compared with neurons growing on laminin. Bar, 100 μm. (bottom) SCG neurons plated on polyornithine and expressing Rlf-CAAX display extensive branching (middle), an effect absent in neurons expressing EGFP-F (left) or inactive Rlf (Rlf-ΔCAT-CAAX; right). Bar, 50 μm. (D) SCG neurons plated on polyornithine were left to express the indicated proteins for 5 h before laminin addition. Pictures were taken after overnight expression. Neurite branching triggered by laminin as observed in cells expressing EGFP-F (top left) is impaired in neurons expressing dominant-negative Ral isoforms (top right and bottom left). (bottom right) A quantitative analysis of neurite branching in five independent laminin-addition experiments (means ± SEM: GFP, 2.19 ± 0.18; RalA28N, 0.84 ± 0.09; RalB28N, 0.95 ± 0.10; **, P < 0.0001). Bar, 30 μm.
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fig3: Ral is activated by laminin and is required for neurite branching promoted by laminin. (A) Phase-contrast pictures of SCG neurons taken 3 h after plating on polyornithine (polorn), polyornithine and laminin (lam), or polyornithine and collagen (coll). Note the visible neurite outgrowth and branching of the laminin-plated neurons compared with cells growing on the other substrates. Bar, 20 μm. (B) SCG neurons were lysed 3 h after plating on the indicated substrates. The amount of RalA-GTP in the lysates was determined by affinity purification with the Ral binding domain of RalBP1 and immunoblotting with anti-RalA antibodies. A representative blot is shown (left), together with the quantification of data of three independent experiments (right; means ± SEM; *, P < 0.005). (C, top) SCG neurons stained for tubulin and photographed 24 h after plating on polyornithine or polyornithine and laminin. Cells plated on polyornithine show limited neurite outgrowth and branching compared with neurons growing on laminin. Bar, 100 μm. (bottom) SCG neurons plated on polyornithine and expressing Rlf-CAAX display extensive branching (middle), an effect absent in neurons expressing EGFP-F (left) or inactive Rlf (Rlf-ΔCAT-CAAX; right). Bar, 50 μm. (D) SCG neurons plated on polyornithine were left to express the indicated proteins for 5 h before laminin addition. Pictures were taken after overnight expression. Neurite branching triggered by laminin as observed in cells expressing EGFP-F (top left) is impaired in neurons expressing dominant-negative Ral isoforms (top right and bottom left). (bottom right) A quantitative analysis of neurite branching in five independent laminin-addition experiments (means ± SEM: GFP, 2.19 ± 0.18; RalA28N, 0.84 ± 0.09; RalB28N, 0.95 ± 0.10; **, P < 0.0001). Bar, 30 μm.
Mentions: Because integrin activation has been implicated in the regulation of neurite outgrowth and branching (Ivins et al., 2000), we tested the influence of different substrates on the levels of Ral activation. SCG neurons were plated on plastic dishes coated with polyornithine only or polyornithine plus either laminin or collagen. 3 h after plating, neuronal morphology varied according to the substrate (Fig. 3 A). SCGs plated on collagen or on polyornithine appeared mainly round with very short processes (Fig. 3 A, top left and bottom). In contrast, cells growing on laminin had longer, branched neurites (Fig. 3 A, top right). To quantify the levels of active Ral under these different conditions, we used a fragment of RalBP1 known to preferentially bind to Ral-GTP as an affinity reagent (Tian et al., 2002). Ral-GTP levels in neurons plated on laminin were approximately fourfold higher than on the other substrates, whereas the total amount of Ral in lysates from different samples was comparable (Fig. 3 B).

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