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Sunday Driver links axonal transport to damage signaling.

Cavalli V, Kujala P, Klumperman J, Goldstein LS - J. Cell Biol. (2005)

Bottom Line: We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex.Finally, we found that injury induces an enhanced interaction between syd and dynactin.Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

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Syd and JNK are transported in both the anterograde and the retrograde pathways. (A) Sciatic nerves were ligated unilaterally at the midpoint and processed for immunofluorescence. APP accumulates primarily on the proximal side of the ligation, whereas phospho-TrkA primarily accumulates on the distal side (arrowheads point to nonspecific staining of the perineurium). Syd and JNK3 accumulate on both proximal and distal sides. White arrows point to ligation site. (B) Ligated and contralateral unligated sciatic nerves were dissected, and extracts were analyzed with the indicated antibodies. Tubulin is used as a loading control. Replicates are shown in the online supplemental material. (C) Sciatic nerve ligation was performed as in A. Nerves were dissected and processed for EM. Proximal and distal sections were stained for syd (15 nm of protein A gold; arrowheads). (D) Total gold particles on an area of 100 μm2 on both proximal and distal axoplasm and an equivalent area on unligated nerve axoplasm were counted. The results are given as the number of gold particles per square micrometer ± SEM. Bars: (A) 100 μm; (C) 200 nm.
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fig3: Syd and JNK are transported in both the anterograde and the retrograde pathways. (A) Sciatic nerves were ligated unilaterally at the midpoint and processed for immunofluorescence. APP accumulates primarily on the proximal side of the ligation, whereas phospho-TrkA primarily accumulates on the distal side (arrowheads point to nonspecific staining of the perineurium). Syd and JNK3 accumulate on both proximal and distal sides. White arrows point to ligation site. (B) Ligated and contralateral unligated sciatic nerves were dissected, and extracts were analyzed with the indicated antibodies. Tubulin is used as a loading control. Replicates are shown in the online supplemental material. (C) Sciatic nerve ligation was performed as in A. Nerves were dissected and processed for EM. Proximal and distal sections were stained for syd (15 nm of protein A gold; arrowheads). (D) Total gold particles on an area of 100 μm2 on both proximal and distal axoplasm and an equivalent area on unligated nerve axoplasm were counted. The results are given as the number of gold particles per square micrometer ± SEM. Bars: (A) 100 μm; (C) 200 nm.

Mentions: The direct interaction between syd and kinesin-I (Bowman et al., 2000) suggests that syd should be transported in axons in the fast anterograde pathway. To test this hypothesis, we performed sciatic nerve ligation experiments. Mouse sciatic nerves were subjected to ligatures for 6 h, and nerve portions proximal or distal to the ligation were analyzed by immunofluorescence microscopy and Western blotting (Fig. 3 A; see Materials and methods). Proteins moving in the fast anterograde axonal transport pathway generally accumulate on the proximal side of a ligation, whereas stationary, slow moving, or nonaxonal proteins remain unchanged. Immunofluorescence of longitudinal sections of a ligated nerve revealed accumulation of APP primarily on the proximal side, as shown previously (Fig. 3 A; Kamal et al., 2000). As a marker for retrograde transport, we performed staining for activated phosphorylated TrkA, which, as expected, accumulated on the distal side (Ehlers et al., 1995; Fig. 3 A). Staining with anti-syd NH2-terminal antibodies revealed that syd accumulates on the proximal side, as expected for a kinesin-I binding protein. We also observed a smaller but significant accumulation on the distal side (Fig. 3 A). Staining with antibodies against JNK3 also revealed accumulation on both the proximal and distal sides (Fig. 3 A), as reported previously (Middlemas et al., 2003).


Sunday Driver links axonal transport to damage signaling.

Cavalli V, Kujala P, Klumperman J, Goldstein LS - J. Cell Biol. (2005)

Syd and JNK are transported in both the anterograde and the retrograde pathways. (A) Sciatic nerves were ligated unilaterally at the midpoint and processed for immunofluorescence. APP accumulates primarily on the proximal side of the ligation, whereas phospho-TrkA primarily accumulates on the distal side (arrowheads point to nonspecific staining of the perineurium). Syd and JNK3 accumulate on both proximal and distal sides. White arrows point to ligation site. (B) Ligated and contralateral unligated sciatic nerves were dissected, and extracts were analyzed with the indicated antibodies. Tubulin is used as a loading control. Replicates are shown in the online supplemental material. (C) Sciatic nerve ligation was performed as in A. Nerves were dissected and processed for EM. Proximal and distal sections were stained for syd (15 nm of protein A gold; arrowheads). (D) Total gold particles on an area of 100 μm2 on both proximal and distal axoplasm and an equivalent area on unligated nerve axoplasm were counted. The results are given as the number of gold particles per square micrometer ± SEM. Bars: (A) 100 μm; (C) 200 nm.
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Related In: Results  -  Collection

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fig3: Syd and JNK are transported in both the anterograde and the retrograde pathways. (A) Sciatic nerves were ligated unilaterally at the midpoint and processed for immunofluorescence. APP accumulates primarily on the proximal side of the ligation, whereas phospho-TrkA primarily accumulates on the distal side (arrowheads point to nonspecific staining of the perineurium). Syd and JNK3 accumulate on both proximal and distal sides. White arrows point to ligation site. (B) Ligated and contralateral unligated sciatic nerves were dissected, and extracts were analyzed with the indicated antibodies. Tubulin is used as a loading control. Replicates are shown in the online supplemental material. (C) Sciatic nerve ligation was performed as in A. Nerves were dissected and processed for EM. Proximal and distal sections were stained for syd (15 nm of protein A gold; arrowheads). (D) Total gold particles on an area of 100 μm2 on both proximal and distal axoplasm and an equivalent area on unligated nerve axoplasm were counted. The results are given as the number of gold particles per square micrometer ± SEM. Bars: (A) 100 μm; (C) 200 nm.
Mentions: The direct interaction between syd and kinesin-I (Bowman et al., 2000) suggests that syd should be transported in axons in the fast anterograde pathway. To test this hypothesis, we performed sciatic nerve ligation experiments. Mouse sciatic nerves were subjected to ligatures for 6 h, and nerve portions proximal or distal to the ligation were analyzed by immunofluorescence microscopy and Western blotting (Fig. 3 A; see Materials and methods). Proteins moving in the fast anterograde axonal transport pathway generally accumulate on the proximal side of a ligation, whereas stationary, slow moving, or nonaxonal proteins remain unchanged. Immunofluorescence of longitudinal sections of a ligated nerve revealed accumulation of APP primarily on the proximal side, as shown previously (Fig. 3 A; Kamal et al., 2000). As a marker for retrograde transport, we performed staining for activated phosphorylated TrkA, which, as expected, accumulated on the distal side (Ehlers et al., 1995; Fig. 3 A). Staining with anti-syd NH2-terminal antibodies revealed that syd accumulates on the proximal side, as expected for a kinesin-I binding protein. We also observed a smaller but significant accumulation on the distal side (Fig. 3 A). Staining with antibodies against JNK3 also revealed accumulation on both the proximal and distal sides (Fig. 3 A), as reported previously (Middlemas et al., 2003).

Bottom Line: We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex.Finally, we found that injury induces an enhanced interaction between syd and dynactin.Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.

Show MeSH
Related in: MedlinePlus