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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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Depletion of endogenous Ral by RNAi decreases branching. Cortical neurons fixed and stained for RalA, RalB, and tubulin 50 h after nucleofection of the indicated siRNA duplexes. (A) Neurons nucleofected with control siRNA display branched neurites visualized by the tubulin staining. RNAi of RalA (B), RalB (C), and both Ral isoforms (D) decreases branching complexity, particularly in major neurites. Bar, 50 μm. (E) Quantitative analysis of branching from major neurites of cortical neurons after RNAi for RalA, RalB, or both Ral isoforms (means ± SEM: control RNAi, 3.39 ± 0.19; RalA RNAi, 1.41 ± 0.11; RalB RNAi, 0.91 ± 0.13; RalA + RalB RNAi, 0.77 ± 0.09; **, P < 0.0001).
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fig2: Depletion of endogenous Ral by RNAi decreases branching. Cortical neurons fixed and stained for RalA, RalB, and tubulin 50 h after nucleofection of the indicated siRNA duplexes. (A) Neurons nucleofected with control siRNA display branched neurites visualized by the tubulin staining. RNAi of RalA (B), RalB (C), and both Ral isoforms (D) decreases branching complexity, particularly in major neurites. Bar, 50 μm. (E) Quantitative analysis of branching from major neurites of cortical neurons after RNAi for RalA, RalB, or both Ral isoforms (means ± SEM: control RNAi, 3.39 ± 0.19; RalA RNAi, 1.41 ± 0.11; RalB RNAi, 0.91 ± 0.13; RalA + RalB RNAi, 0.77 ± 0.09; **, P < 0.0001).

Mentions: To gain further evidence for a role of Ral in neurite branching, we depleted cortical neurons of RalA, RalB, or both by RNAi. Small interfering RNA (siRNA) duplexes against RalA, RalB, or both were introduced in freshly dissociated cortical neurons by nucleofection. Cells were plated, fixed, and stained 50 h later for the Ral isoforms and for tubulin to visualize microtubules. Control cells nucleofected with a scramble siRNA duplex directed against GFP showed a typical polarized morphology, with short neurites and a major process extending from the cell body. All these neurites display a certain branching complexity, as visualized by the microtubule staining (Fig. 2 A, right). In these cells, endogenous RalA is abundant and equally distributed in all neurites (Fig. 2 A, left), whereas RalB appears to be especially concentrated in the terminal portion of major neurites (Fig. 2 A, middle). Neurons transfected with the siRNA against RalA displayed a general decrease in RalA immunoreactivity, whereas RalB staining remained (Fig. 2 B, left and middle). The opposite effect was observed with the siRNA against RalB (Fig. 2 C, left and middle). Importantly, in both cases, neurons were characterized by a simpler branching morphology compared with controls (Fig. 2, compare the tubulin stainings in B and C [right] with those in A [right]). Finally, RNAi of both Ral isoforms also led to a substantial decrease in neurite branching (Fig. 2 D).


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

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

Depletion of endogenous Ral by RNAi decreases branching. Cortical neurons fixed and stained for RalA, RalB, and tubulin 50 h after nucleofection of the indicated siRNA duplexes. (A) Neurons nucleofected with control siRNA display branched neurites visualized by the tubulin staining. RNAi of RalA (B), RalB (C), and both Ral isoforms (D) decreases branching complexity, particularly in major neurites. Bar, 50 μm. (E) Quantitative analysis of branching from major neurites of cortical neurons after RNAi for RalA, RalB, or both Ral isoforms (means ± SEM: control RNAi, 3.39 ± 0.19; RalA RNAi, 1.41 ± 0.11; RalB RNAi, 0.91 ± 0.13; RalA + RalB RNAi, 0.77 ± 0.09; **, P < 0.0001).
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getmorefigures.php?uid=PMC2171284&req=5

fig2: Depletion of endogenous Ral by RNAi decreases branching. Cortical neurons fixed and stained for RalA, RalB, and tubulin 50 h after nucleofection of the indicated siRNA duplexes. (A) Neurons nucleofected with control siRNA display branched neurites visualized by the tubulin staining. RNAi of RalA (B), RalB (C), and both Ral isoforms (D) decreases branching complexity, particularly in major neurites. Bar, 50 μm. (E) Quantitative analysis of branching from major neurites of cortical neurons after RNAi for RalA, RalB, or both Ral isoforms (means ± SEM: control RNAi, 3.39 ± 0.19; RalA RNAi, 1.41 ± 0.11; RalB RNAi, 0.91 ± 0.13; RalA + RalB RNAi, 0.77 ± 0.09; **, P < 0.0001).
Mentions: To gain further evidence for a role of Ral in neurite branching, we depleted cortical neurons of RalA, RalB, or both by RNAi. Small interfering RNA (siRNA) duplexes against RalA, RalB, or both were introduced in freshly dissociated cortical neurons by nucleofection. Cells were plated, fixed, and stained 50 h later for the Ral isoforms and for tubulin to visualize microtubules. Control cells nucleofected with a scramble siRNA duplex directed against GFP showed a typical polarized morphology, with short neurites and a major process extending from the cell body. All these neurites display a certain branching complexity, as visualized by the microtubule staining (Fig. 2 A, right). In these cells, endogenous RalA is abundant and equally distributed in all neurites (Fig. 2 A, left), whereas RalB appears to be especially concentrated in the terminal portion of major neurites (Fig. 2 A, middle). Neurons transfected with the siRNA against RalA displayed a general decrease in RalA immunoreactivity, whereas RalB staining remained (Fig. 2 B, left and middle). The opposite effect was observed with the siRNA against RalB (Fig. 2 C, left and middle). Importantly, in both cases, neurons were characterized by a simpler branching morphology compared with controls (Fig. 2, compare the tubulin stainings in B and C [right] with those in A [right]). Finally, RNAi of both Ral isoforms also led to a substantial decrease in neurite branching (Fig. 2 D).

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