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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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Branching is mediated by distinct RalA and -B effectors. (A) An SCG neuron expressing EGFP-F 24 h after microinjection. (B) SCG neurons expressing the indicated proteins visualized by anti-myc immunostaining. Active RalA (RalA72L) increases branching, but this effect is particularly impaired in cells expressing active RalA unable to interact with the exocyst complex (RalA72LD49E). The increased branching caused by active RalB (RalB23V) is inhibited in neurons expressing active RalB unable to associate with PLD (RalB23VΔN11). Other RalB effector domain mutants retain branch-promoting activity. Bar, 100 μm. (C) Quantitative analysis of branching for control cells expressing EGFP-F, active RalA or -B, and their effector mutants (means ± SEM: GFP, 3.44 ± 0.13; RalA72L, 9.34 ± 0.83; RalA72LΔN11, 7.37 ± 0.45; RalA72LD49E, 4.68 ± 0.43; RalA72LD49N, 8.43 ± 0.59; RalB23V, 7.09 ± 0.42; RalB23VΔN11, 5.26 ± 0.34; RalB23VD49E, 8.62 ± 0.48; RalB23VD49N, 8.83 ± 0.47; *, P < 0.006; **, P < 0.0001; #, P < 0.03; ##, P < 0.0001). (D) Quantitative analysis of branching in cells expressing active RalA or -B and incubated with 0.4% 1-butanol or 2-butanol. PLD inhibition by 1-butanol abolishes the effect of RalB on branching while partially inhibiting the increased branching caused by RalA. Control treatment with 2-butanol does not affect the branch-promoting activity of either Ral isoform (means ± SEM: RalA72L, 9.34 ± 0.83; RalA72L + 1-but, 5.78 ± 0.62; RalA72L + 2-but, 9.12 ± 0.08; RalB23V, 7.09 ± 0.42; RalB23 + 1-but, 3.34 ± 0.47; RalB23V + 2-but, 7.04 ± 0.66; **, P < 0.0001).
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fig8: Branching is mediated by distinct RalA and -B effectors. (A) An SCG neuron expressing EGFP-F 24 h after microinjection. (B) SCG neurons expressing the indicated proteins visualized by anti-myc immunostaining. Active RalA (RalA72L) increases branching, but this effect is particularly impaired in cells expressing active RalA unable to interact with the exocyst complex (RalA72LD49E). The increased branching caused by active RalB (RalB23V) is inhibited in neurons expressing active RalB unable to associate with PLD (RalB23VΔN11). Other RalB effector domain mutants retain branch-promoting activity. Bar, 100 μm. (C) Quantitative analysis of branching for control cells expressing EGFP-F, active RalA or -B, and their effector mutants (means ± SEM: GFP, 3.44 ± 0.13; RalA72L, 9.34 ± 0.83; RalA72LΔN11, 7.37 ± 0.45; RalA72LD49E, 4.68 ± 0.43; RalA72LD49N, 8.43 ± 0.59; RalB23V, 7.09 ± 0.42; RalB23VΔN11, 5.26 ± 0.34; RalB23VD49E, 8.62 ± 0.48; RalB23VD49N, 8.83 ± 0.47; *, P < 0.006; **, P < 0.0001; #, P < 0.03; ##, P < 0.0001). (D) Quantitative analysis of branching in cells expressing active RalA or -B and incubated with 0.4% 1-butanol or 2-butanol. PLD inhibition by 1-butanol abolishes the effect of RalB on branching while partially inhibiting the increased branching caused by RalA. Control treatment with 2-butanol does not affect the branch-promoting activity of either Ral isoform (means ± SEM: RalA72L, 9.34 ± 0.83; RalA72L + 1-but, 5.78 ± 0.62; RalA72L + 2-but, 9.12 ± 0.08; RalB23V, 7.09 ± 0.42; RalB23 + 1-but, 3.34 ± 0.47; RalB23V + 2-but, 7.04 ± 0.66; **, P < 0.0001).

Mentions: Along with published data from others, the results in Fig. 7 raise the possibility that RalA and -B preferentially activate different downstream pathways (Chien and White, 2003; Shipitsin and Feig, 2004). To determine whether multiple effectors are responsible for Ral-induced neurite branching, we used Ral mutants previously identified on the basis of their loss of binding affinity for specific effector proteins. In particular, we used myc-tagged constitutively active RalA and -B isoforms unable to bind the Sec5 and Exo84 exocyst complex subunits (RalA72LD49E and -B23VD49E; Moskalenko et al., 2002, 2003) or selectively uncoupled from RalBP1 (RalA72LD49N and -B23VD49N; Jullien-Flores et al., 2000). We also used active Ral mutants lacking the NH2-terminal 11 amino acids (RalA72LΔN11 and -B23VΔN11), which are required for association with PLD (Jiang et al., 1995). SCG neurons plated on laminin were microinjected either with EGFP-F as control or with active RalA, RalB, or the Ral effector domain mutants. Cells were fixed 24 h after injection and stained with anti-myc antibodies. As expected, active RalA and -B increased branching compared with EGFP-F (Fig. 8, A–C, compare EGFP-F, RalA72L, and RalB23V). The RalA mutant unable to interact with PLD showed a small but significant reduction in branch-promoting ability, whereas the mutant uncoupled from RalBP1 was as effective as active RalA (Fig. 8, B and C, compare RalA72L with RalA72LΔN11 and RalA72LD49N). Remarkably, however, preventing the interaction of active RalA with the exocyst complex dramatically inhibited its ability to promote branching (Fig. 8, B and C, compare RalA72L with RalA72LD49E). Interestingly, the RalB mutants unable to interact with either the exocyst complex or RalBP1 promoted branching similarly (if not better) to active RalB (Fig. 8, B and C, compare RalB23V with RalB23VD49E and RalB23VD49N). In contrast, the RalB mutant unable to interact with PLD displayed a significant decrease in branch-promoting activity (Fig. 8, A–C, compare RalB23VΔN11 with RalB23V and EGFP). To provide additional support for a role for PLD in Ral-induced branching, SCG neurons were incubated with 0.4% 1-butanol, a PLD inhibitor (Morris et al., 1997), after injection with active Ral. This treatment significantly abolished RalA- and completely blocked RalB-dependent increases in branching, whereas incubation with 0.4% 2-butanol, as a control, had no effect (Fig. 8 D). Together, these results indicate that Ral may increase neurite branching through the exocyst complex and PLD.


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

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

Branching is mediated by distinct RalA and -B effectors. (A) An SCG neuron expressing EGFP-F 24 h after microinjection. (B) SCG neurons expressing the indicated proteins visualized by anti-myc immunostaining. Active RalA (RalA72L) increases branching, but this effect is particularly impaired in cells expressing active RalA unable to interact with the exocyst complex (RalA72LD49E). The increased branching caused by active RalB (RalB23V) is inhibited in neurons expressing active RalB unable to associate with PLD (RalB23VΔN11). Other RalB effector domain mutants retain branch-promoting activity. Bar, 100 μm. (C) Quantitative analysis of branching for control cells expressing EGFP-F, active RalA or -B, and their effector mutants (means ± SEM: GFP, 3.44 ± 0.13; RalA72L, 9.34 ± 0.83; RalA72LΔN11, 7.37 ± 0.45; RalA72LD49E, 4.68 ± 0.43; RalA72LD49N, 8.43 ± 0.59; RalB23V, 7.09 ± 0.42; RalB23VΔN11, 5.26 ± 0.34; RalB23VD49E, 8.62 ± 0.48; RalB23VD49N, 8.83 ± 0.47; *, P < 0.006; **, P < 0.0001; #, P < 0.03; ##, P < 0.0001). (D) Quantitative analysis of branching in cells expressing active RalA or -B and incubated with 0.4% 1-butanol or 2-butanol. PLD inhibition by 1-butanol abolishes the effect of RalB on branching while partially inhibiting the increased branching caused by RalA. Control treatment with 2-butanol does not affect the branch-promoting activity of either Ral isoform (means ± SEM: RalA72L, 9.34 ± 0.83; RalA72L + 1-but, 5.78 ± 0.62; RalA72L + 2-but, 9.12 ± 0.08; RalB23V, 7.09 ± 0.42; RalB23 + 1-but, 3.34 ± 0.47; RalB23V + 2-but, 7.04 ± 0.66; **, P < 0.0001).
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fig8: Branching is mediated by distinct RalA and -B effectors. (A) An SCG neuron expressing EGFP-F 24 h after microinjection. (B) SCG neurons expressing the indicated proteins visualized by anti-myc immunostaining. Active RalA (RalA72L) increases branching, but this effect is particularly impaired in cells expressing active RalA unable to interact with the exocyst complex (RalA72LD49E). The increased branching caused by active RalB (RalB23V) is inhibited in neurons expressing active RalB unable to associate with PLD (RalB23VΔN11). Other RalB effector domain mutants retain branch-promoting activity. Bar, 100 μm. (C) Quantitative analysis of branching for control cells expressing EGFP-F, active RalA or -B, and their effector mutants (means ± SEM: GFP, 3.44 ± 0.13; RalA72L, 9.34 ± 0.83; RalA72LΔN11, 7.37 ± 0.45; RalA72LD49E, 4.68 ± 0.43; RalA72LD49N, 8.43 ± 0.59; RalB23V, 7.09 ± 0.42; RalB23VΔN11, 5.26 ± 0.34; RalB23VD49E, 8.62 ± 0.48; RalB23VD49N, 8.83 ± 0.47; *, P < 0.006; **, P < 0.0001; #, P < 0.03; ##, P < 0.0001). (D) Quantitative analysis of branching in cells expressing active RalA or -B and incubated with 0.4% 1-butanol or 2-butanol. PLD inhibition by 1-butanol abolishes the effect of RalB on branching while partially inhibiting the increased branching caused by RalA. Control treatment with 2-butanol does not affect the branch-promoting activity of either Ral isoform (means ± SEM: RalA72L, 9.34 ± 0.83; RalA72L + 1-but, 5.78 ± 0.62; RalA72L + 2-but, 9.12 ± 0.08; RalB23V, 7.09 ± 0.42; RalB23 + 1-but, 3.34 ± 0.47; RalB23V + 2-but, 7.04 ± 0.66; **, P < 0.0001).
Mentions: Along with published data from others, the results in Fig. 7 raise the possibility that RalA and -B preferentially activate different downstream pathways (Chien and White, 2003; Shipitsin and Feig, 2004). To determine whether multiple effectors are responsible for Ral-induced neurite branching, we used Ral mutants previously identified on the basis of their loss of binding affinity for specific effector proteins. In particular, we used myc-tagged constitutively active RalA and -B isoforms unable to bind the Sec5 and Exo84 exocyst complex subunits (RalA72LD49E and -B23VD49E; Moskalenko et al., 2002, 2003) or selectively uncoupled from RalBP1 (RalA72LD49N and -B23VD49N; Jullien-Flores et al., 2000). We also used active Ral mutants lacking the NH2-terminal 11 amino acids (RalA72LΔN11 and -B23VΔN11), which are required for association with PLD (Jiang et al., 1995). SCG neurons plated on laminin were microinjected either with EGFP-F as control or with active RalA, RalB, or the Ral effector domain mutants. Cells were fixed 24 h after injection and stained with anti-myc antibodies. As expected, active RalA and -B increased branching compared with EGFP-F (Fig. 8, A–C, compare EGFP-F, RalA72L, and RalB23V). The RalA mutant unable to interact with PLD showed a small but significant reduction in branch-promoting ability, whereas the mutant uncoupled from RalBP1 was as effective as active RalA (Fig. 8, B and C, compare RalA72L with RalA72LΔN11 and RalA72LD49N). Remarkably, however, preventing the interaction of active RalA with the exocyst complex dramatically inhibited its ability to promote branching (Fig. 8, B and C, compare RalA72L with RalA72LD49E). Interestingly, the RalB mutants unable to interact with either the exocyst complex or RalBP1 promoted branching similarly (if not better) to active RalB (Fig. 8, B and C, compare RalB23V with RalB23VD49E and RalB23VD49N). In contrast, the RalB mutant unable to interact with PLD displayed a significant decrease in branch-promoting activity (Fig. 8, A–C, compare RalB23VΔN11 with RalB23V and EGFP). To provide additional support for a role for PLD in Ral-induced branching, SCG neurons were incubated with 0.4% 1-butanol, a PLD inhibitor (Morris et al., 1997), after injection with active Ral. This treatment significantly abolished RalA- and completely blocked RalB-dependent increases in branching, whereas incubation with 0.4% 2-butanol, as a control, had no effect (Fig. 8 D). Together, these results indicate that Ral may increase neurite branching through the exocyst complex and PLD.

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