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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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Active Ral increases neurite branching. SCG neurons (A) and cortical neurons (B) expressing EGFP-F or the indicated proteins visualized by anti-myc (for Ral) or anti-HA (for Rlf-CAAX) immunostaining. Pictures were taken 20 h after transfection. Constitutively active RalA (RalA72L) and -B (RalB23V) or the active Ral-GEF Rlf-CAAX increase neurite branching, whereas dominant-negative Ral (RalA28N and -B28N) decreases branching. SCG neurons were plated on laminin-coated dishes, whereas cortical neurons were grown on polyornithine. Bars: (A) 100 μm; (B) 50 μm. (C and D) Quantitative analysis of neurite morphology. Branch density per 100 μm of neurite length (C) is highly increased with Rlf-CAAX and constitutively active Ral and decreased with dominant-negative Ral (means ± SEM: GFP, 0.65 ± 0.03; Rlf-CAAX, 1.45 ± 0.14; RalA72L, 1.17 ± 0.12; RalB23V, 1.62 ± 0.09; RalA28N, 0.52 ± 0.03; RalB28N, 0.48 ± 0.03; **, P < 0.0001; *, P < 0.01). In contrast, average axon length (D) does not vary significantly, except for Ral A (means ± SEM: GFP, 261.62 ± 8.65; Rlf-CAAX, 266.41 ± 16.92; RalA72L, 402.54 ± 18.57; RalB23V, 274.53 ± 9.01; RalA28N, 216.89 ± 7.92; RalB28N, 238.74 ± 14.52; **, P < 0.0001). (E and F) Quantitative analysis of the number of primary neurites emerging from the cell body and containing microtubules in SCG neurons (means ± SEM: GFP, 3.38 ± 0.06; Rlf-CAAX, 3.20 ± 0.07; RalA72L, 4.25 ± 0.17; RalB23V, 4.03 ± 0.11; RalA28N, 2.91 ± 0.10; RalB28N, 2.93 ± 0.10; **, P < 0.0001) and cortical neurons (means ± SEM: GFP, 5.95 ± 0.23; Rlf-CAAX, 7.33 ± 0.18; RalA72L, 7.67 ± 0.26; RalB23V, 8.42 ± 0.25; RalA28N, 3.98 ± 0.20; RalB28N, 3.78 ± 0.18; **, P < 0.0001).
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fig1: Active Ral increases neurite branching. SCG neurons (A) and cortical neurons (B) expressing EGFP-F or the indicated proteins visualized by anti-myc (for Ral) or anti-HA (for Rlf-CAAX) immunostaining. Pictures were taken 20 h after transfection. Constitutively active RalA (RalA72L) and -B (RalB23V) or the active Ral-GEF Rlf-CAAX increase neurite branching, whereas dominant-negative Ral (RalA28N and -B28N) decreases branching. SCG neurons were plated on laminin-coated dishes, whereas cortical neurons were grown on polyornithine. Bars: (A) 100 μm; (B) 50 μm. (C and D) Quantitative analysis of neurite morphology. Branch density per 100 μm of neurite length (C) is highly increased with Rlf-CAAX and constitutively active Ral and decreased with dominant-negative Ral (means ± SEM: GFP, 0.65 ± 0.03; Rlf-CAAX, 1.45 ± 0.14; RalA72L, 1.17 ± 0.12; RalB23V, 1.62 ± 0.09; RalA28N, 0.52 ± 0.03; RalB28N, 0.48 ± 0.03; **, P < 0.0001; *, P < 0.01). In contrast, average axon length (D) does not vary significantly, except for Ral A (means ± SEM: GFP, 261.62 ± 8.65; Rlf-CAAX, 266.41 ± 16.92; RalA72L, 402.54 ± 18.57; RalB23V, 274.53 ± 9.01; RalA28N, 216.89 ± 7.92; RalB28N, 238.74 ± 14.52; **, P < 0.0001). (E and F) Quantitative analysis of the number of primary neurites emerging from the cell body and containing microtubules in SCG neurons (means ± SEM: GFP, 3.38 ± 0.06; Rlf-CAAX, 3.20 ± 0.07; RalA72L, 4.25 ± 0.17; RalB23V, 4.03 ± 0.11; RalA28N, 2.91 ± 0.10; RalB28N, 2.93 ± 0.10; **, P < 0.0001) and cortical neurons (means ± SEM: GFP, 5.95 ± 0.23; Rlf-CAAX, 7.33 ± 0.18; RalA72L, 7.67 ± 0.26; RalB23V, 8.42 ± 0.25; RalA28N, 3.98 ± 0.20; RalB28N, 3.78 ± 0.18; **, P < 0.0001).

Mentions: To determine whether Ral GTPases are able to influence neuronal morphology, we microinjected sympathetic superior cervical ganglia (SCG) neurons (Fig. 1 A) or nucleofected cortical neurons (Fig. 1 B) with constitutively active Ral (RalA72L and -B23V), dominant-negative Ral (RalA28N and -B28N), or Rlf-CAAX, a constitutively active Ral-GEF (Wolthuis et al., 1997). SCG neurons were microinjected 2 h after plating, when only short neurites growing from somata were visible, whereas cortical neurons were nucleofected before plating. A farnesylated version of EGFP (EGFP-F) was used to visualize neuronal morphology. Protein expression was confirmed by staining cells with anti-myc or anti-HA antibodies for Ral and Rlf, respectively (Fig. 1, A and B). SCG neurons tend to extend only long processes, whereas cortical neurons adopt a typical polarized morphology with several short neurites and a single longer, axonlike process. Expression of constitutively active Ral isoforms or activation of endogenous Ral with Rlf-CAAX in both types of neuron enhanced cell body spreading and branching complexity (Fig. 1, A and B, top right and middle). Quantification revealed a dramatic increase of branch density, ranging from 80 to 150%, in cells expressing constitutively active Ral or Rlf-CAAX compared with control cells expressing EGFP (Fig. 1 C), whereas cells expressing dominant-negative Ral were characterized by a decreased number of branches and a significant (20–25%) decrease in branch density (Fig. 1, A and B [bottom] and C). Rlf-ΔCAT-CAAX, a Rlf-CAAX control construct lacking catalytic activity (Wolthuis et al., 1997), did not affect branching (unpublished data). The average axon length did not change significantly when expressing Rlf-CAAX (Fig. 1 D), indicating that activation of endogenous Ral proteins induces a specific effect on branching. Interestingly, constitutively activated and dominant-negative RalA, but not -B, also had an effect on axon length in SCG neurons (Fig. 1 D), whereas both isoforms had an effect on the number of primary neurites per cell in both SCG and cortical neurons (Fig. 1, E and F). These results suggest that Ral may play other roles in addition to branching.


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

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

Active Ral increases neurite branching. SCG neurons (A) and cortical neurons (B) expressing EGFP-F or the indicated proteins visualized by anti-myc (for Ral) or anti-HA (for Rlf-CAAX) immunostaining. Pictures were taken 20 h after transfection. Constitutively active RalA (RalA72L) and -B (RalB23V) or the active Ral-GEF Rlf-CAAX increase neurite branching, whereas dominant-negative Ral (RalA28N and -B28N) decreases branching. SCG neurons were plated on laminin-coated dishes, whereas cortical neurons were grown on polyornithine. Bars: (A) 100 μm; (B) 50 μm. (C and D) Quantitative analysis of neurite morphology. Branch density per 100 μm of neurite length (C) is highly increased with Rlf-CAAX and constitutively active Ral and decreased with dominant-negative Ral (means ± SEM: GFP, 0.65 ± 0.03; Rlf-CAAX, 1.45 ± 0.14; RalA72L, 1.17 ± 0.12; RalB23V, 1.62 ± 0.09; RalA28N, 0.52 ± 0.03; RalB28N, 0.48 ± 0.03; **, P < 0.0001; *, P < 0.01). In contrast, average axon length (D) does not vary significantly, except for Ral A (means ± SEM: GFP, 261.62 ± 8.65; Rlf-CAAX, 266.41 ± 16.92; RalA72L, 402.54 ± 18.57; RalB23V, 274.53 ± 9.01; RalA28N, 216.89 ± 7.92; RalB28N, 238.74 ± 14.52; **, P < 0.0001). (E and F) Quantitative analysis of the number of primary neurites emerging from the cell body and containing microtubules in SCG neurons (means ± SEM: GFP, 3.38 ± 0.06; Rlf-CAAX, 3.20 ± 0.07; RalA72L, 4.25 ± 0.17; RalB23V, 4.03 ± 0.11; RalA28N, 2.91 ± 0.10; RalB28N, 2.93 ± 0.10; **, P < 0.0001) and cortical neurons (means ± SEM: GFP, 5.95 ± 0.23; Rlf-CAAX, 7.33 ± 0.18; RalA72L, 7.67 ± 0.26; RalB23V, 8.42 ± 0.25; RalA28N, 3.98 ± 0.20; RalB28N, 3.78 ± 0.18; **, P < 0.0001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171284&req=5

fig1: Active Ral increases neurite branching. SCG neurons (A) and cortical neurons (B) expressing EGFP-F or the indicated proteins visualized by anti-myc (for Ral) or anti-HA (for Rlf-CAAX) immunostaining. Pictures were taken 20 h after transfection. Constitutively active RalA (RalA72L) and -B (RalB23V) or the active Ral-GEF Rlf-CAAX increase neurite branching, whereas dominant-negative Ral (RalA28N and -B28N) decreases branching. SCG neurons were plated on laminin-coated dishes, whereas cortical neurons were grown on polyornithine. Bars: (A) 100 μm; (B) 50 μm. (C and D) Quantitative analysis of neurite morphology. Branch density per 100 μm of neurite length (C) is highly increased with Rlf-CAAX and constitutively active Ral and decreased with dominant-negative Ral (means ± SEM: GFP, 0.65 ± 0.03; Rlf-CAAX, 1.45 ± 0.14; RalA72L, 1.17 ± 0.12; RalB23V, 1.62 ± 0.09; RalA28N, 0.52 ± 0.03; RalB28N, 0.48 ± 0.03; **, P < 0.0001; *, P < 0.01). In contrast, average axon length (D) does not vary significantly, except for Ral A (means ± SEM: GFP, 261.62 ± 8.65; Rlf-CAAX, 266.41 ± 16.92; RalA72L, 402.54 ± 18.57; RalB23V, 274.53 ± 9.01; RalA28N, 216.89 ± 7.92; RalB28N, 238.74 ± 14.52; **, P < 0.0001). (E and F) Quantitative analysis of the number of primary neurites emerging from the cell body and containing microtubules in SCG neurons (means ± SEM: GFP, 3.38 ± 0.06; Rlf-CAAX, 3.20 ± 0.07; RalA72L, 4.25 ± 0.17; RalB23V, 4.03 ± 0.11; RalA28N, 2.91 ± 0.10; RalB28N, 2.93 ± 0.10; **, P < 0.0001) and cortical neurons (means ± SEM: GFP, 5.95 ± 0.23; Rlf-CAAX, 7.33 ± 0.18; RalA72L, 7.67 ± 0.26; RalB23V, 8.42 ± 0.25; RalA28N, 3.98 ± 0.20; RalB28N, 3.78 ± 0.18; **, P < 0.0001).
Mentions: To determine whether Ral GTPases are able to influence neuronal morphology, we microinjected sympathetic superior cervical ganglia (SCG) neurons (Fig. 1 A) or nucleofected cortical neurons (Fig. 1 B) with constitutively active Ral (RalA72L and -B23V), dominant-negative Ral (RalA28N and -B28N), or Rlf-CAAX, a constitutively active Ral-GEF (Wolthuis et al., 1997). SCG neurons were microinjected 2 h after plating, when only short neurites growing from somata were visible, whereas cortical neurons were nucleofected before plating. A farnesylated version of EGFP (EGFP-F) was used to visualize neuronal morphology. Protein expression was confirmed by staining cells with anti-myc or anti-HA antibodies for Ral and Rlf, respectively (Fig. 1, A and B). SCG neurons tend to extend only long processes, whereas cortical neurons adopt a typical polarized morphology with several short neurites and a single longer, axonlike process. Expression of constitutively active Ral isoforms or activation of endogenous Ral with Rlf-CAAX in both types of neuron enhanced cell body spreading and branching complexity (Fig. 1, A and B, top right and middle). Quantification revealed a dramatic increase of branch density, ranging from 80 to 150%, in cells expressing constitutively active Ral or Rlf-CAAX compared with control cells expressing EGFP (Fig. 1 C), whereas cells expressing dominant-negative Ral were characterized by a decreased number of branches and a significant (20–25%) decrease in branch density (Fig. 1, A and B [bottom] and C). Rlf-ΔCAT-CAAX, a Rlf-CAAX control construct lacking catalytic activity (Wolthuis et al., 1997), did not affect branching (unpublished data). The average axon length did not change significantly when expressing Rlf-CAAX (Fig. 1 D), indicating that activation of endogenous Ral proteins induces a specific effect on branching. Interestingly, constitutively activated and dominant-negative RalA, but not -B, also had an effect on axon length in SCG neurons (Fig. 1 D), whereas both isoforms had an effect on the number of primary neurites per cell in both SCG and cortical neurons (Fig. 1, E and F). These results suggest that Ral may play other roles in addition to 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