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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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Branches induced by Ral activation depend on F-actin. (A, left) Confocal image of an SCG neuron plated on polyornithine expressing Rlf-CAAX (red) and stained for tubulin (green). The middle and right panels are enlargements of the boxed area in the left panel. Longer branches are positive for tubulin (arrowheads). Bar, 20 μm. (B) In Rlf-CAAX–expressing neurons, short branches contain actin filaments visualized by fluorescent phalloidin (green) but not tubulin (white, blue in the merge panel). Note the accumulation of Rlf-CAAX (red) in discrete domains along branches (arrowheads). Bar, 10 μm. (C) Representative images of SCG neurons plated on polyornithine, expressing Rlf-CAAX, and treated with the indicated amounts of cytochalasin D. Increasing drug doses led to a progressive block of Ral-dependent branching. Bar, 50 μm.
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fig4: Branches induced by Ral activation depend on F-actin. (A, left) Confocal image of an SCG neuron plated on polyornithine expressing Rlf-CAAX (red) and stained for tubulin (green). The middle and right panels are enlargements of the boxed area in the left panel. Longer branches are positive for tubulin (arrowheads). Bar, 20 μm. (B) In Rlf-CAAX–expressing neurons, short branches contain actin filaments visualized by fluorescent phalloidin (green) but not tubulin (white, blue in the merge panel). Note the accumulation of Rlf-CAAX (red) in discrete domains along branches (arrowheads). Bar, 10 μm. (C) Representative images of SCG neurons plated on polyornithine, expressing Rlf-CAAX, and treated with the indicated amounts of cytochalasin D. Increasing drug doses led to a progressive block of Ral-dependent branching. Bar, 50 μm.

Mentions: Neurite branching requires reorganization of the cytoskeleton and is generally initiated by the appearance of motile filopodia in response to extracellular cues (Gallo and Letourneau, 2004). We stained the Ral-induced branches with fluorescent phalloidin to visualize actin filaments and an anti-tubulin antibody to reveal microtubules. In SCG neurons expressing Rlf-CAAX, phalloidin labeled filopodia, short nascent branches, and long branches, but microtubules could be detected only in longer branches (Fig. 4, A and B). Notably, Rlf-CAAX appeared to be concentrated in puncta along neurites and sprouting branches and was often present at branch points (Fig. 4 B, arrowheads). SCG neurons expressing constitutively active Ral showed clear filopodial sprouting also from cell bodies (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200507061/DC1). Treatment of neurons with cytochalasin D, an inhibitor of actin polymerization, blocked filopodial sprouting and branching in a dose-dependent manner (Fig. 4 C). These observations suggest that active Ral causes branching by promoting the formation of filopodia and then allowing microtubules to invade the branch, consistent with previous studies on branch initiation (Dent et al., 2003; Gallo and Letourneau, 2004).


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

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

Branches induced by Ral activation depend on F-actin. (A, left) Confocal image of an SCG neuron plated on polyornithine expressing Rlf-CAAX (red) and stained for tubulin (green). The middle and right panels are enlargements of the boxed area in the left panel. Longer branches are positive for tubulin (arrowheads). Bar, 20 μm. (B) In Rlf-CAAX–expressing neurons, short branches contain actin filaments visualized by fluorescent phalloidin (green) but not tubulin (white, blue in the merge panel). Note the accumulation of Rlf-CAAX (red) in discrete domains along branches (arrowheads). Bar, 10 μm. (C) Representative images of SCG neurons plated on polyornithine, expressing Rlf-CAAX, and treated with the indicated amounts of cytochalasin D. Increasing drug doses led to a progressive block of Ral-dependent branching. Bar, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171284&req=5

fig4: Branches induced by Ral activation depend on F-actin. (A, left) Confocal image of an SCG neuron plated on polyornithine expressing Rlf-CAAX (red) and stained for tubulin (green). The middle and right panels are enlargements of the boxed area in the left panel. Longer branches are positive for tubulin (arrowheads). Bar, 20 μm. (B) In Rlf-CAAX–expressing neurons, short branches contain actin filaments visualized by fluorescent phalloidin (green) but not tubulin (white, blue in the merge panel). Note the accumulation of Rlf-CAAX (red) in discrete domains along branches (arrowheads). Bar, 10 μm. (C) Representative images of SCG neurons plated on polyornithine, expressing Rlf-CAAX, and treated with the indicated amounts of cytochalasin D. Increasing drug doses led to a progressive block of Ral-dependent branching. Bar, 50 μm.
Mentions: Neurite branching requires reorganization of the cytoskeleton and is generally initiated by the appearance of motile filopodia in response to extracellular cues (Gallo and Letourneau, 2004). We stained the Ral-induced branches with fluorescent phalloidin to visualize actin filaments and an anti-tubulin antibody to reveal microtubules. In SCG neurons expressing Rlf-CAAX, phalloidin labeled filopodia, short nascent branches, and long branches, but microtubules could be detected only in longer branches (Fig. 4, A and B). Notably, Rlf-CAAX appeared to be concentrated in puncta along neurites and sprouting branches and was often present at branch points (Fig. 4 B, arrowheads). SCG neurons expressing constitutively active Ral showed clear filopodial sprouting also from cell bodies (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200507061/DC1). Treatment of neurons with cytochalasin D, an inhibitor of actin polymerization, blocked filopodial sprouting and branching in a dose-dependent manner (Fig. 4 C). These observations suggest that active Ral causes branching by promoting the formation of filopodia and then allowing microtubules to invade the branch, consistent with previous studies on branch initiation (Dent et al., 2003; Gallo and Letourneau, 2004).

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