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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 association with the dynactin complex is increased by injury. The dynactin complex was immunodepleted from membrane-bound material of ligated or unligated sciatic nerves. For dynactin depletion, both p150Glued and p50 antibodies were used, whereas for mock depletion a GFP antibody was used. (A) SDS-PAGE analysis of immunodepletion using dynactin (dyn) or GFP antibodies. Note that input (IN) and immunoprecipitated material (IP) are from equivalent exposures, except for blots with syd and P-JNK antibodies, which required higher exposure times. (B) The unbound (nonimmunoprecipitated) material was sedimented on 5–20% linear sucrose gradients. Fractions were resolved by SDS-PAGE and Western blots were performed using syd, p150Glued, DIC, and KIF5C antibodies. Quantification is expressed as a percentage of total ± SEM (n = 3). Dynactin depletion (closed symbols) and mock GFP depletion (open symbols) is shown. A representative Western blot is shown in the online supplemental material.
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fig8: Syd association with the dynactin complex is increased by injury. The dynactin complex was immunodepleted from membrane-bound material of ligated or unligated sciatic nerves. For dynactin depletion, both p150Glued and p50 antibodies were used, whereas for mock depletion a GFP antibody was used. (A) SDS-PAGE analysis of immunodepletion using dynactin (dyn) or GFP antibodies. Note that input (IN) and immunoprecipitated material (IP) are from equivalent exposures, except for blots with syd and P-JNK antibodies, which required higher exposure times. (B) The unbound (nonimmunoprecipitated) material was sedimented on 5–20% linear sucrose gradients. Fractions were resolved by SDS-PAGE and Western blots were performed using syd, p150Glued, DIC, and KIF5C antibodies. Quantification is expressed as a percentage of total ± SEM (n = 3). Dynactin depletion (closed symbols) and mock GFP depletion (open symbols) is shown. A representative Western blot is shown in the online supplemental material.

Mentions: Although generally increased axonal transport following injury has been demonstrated (Curtis et al., 1998), evidence for injury-induced transport of signaling molecules other than neurotrophins does not exist (Delcroix et al., 2003a). Thus, we tested if sciatic nerve injury results in an increase of syd association with the dynactin complex, which might account for a switch to retrograde transport. By performing sucrose density sedimentation analysis, we observed that the soluble pool of syd is found exclusively at ∼8S (unpublished data), whereas the membrane-associated pool is found in two peaks, ∼8S and ∼17S. To test if the syd peak at ∼17S reports an interaction with dynactin, we performed sucrose density gradient sedimentation analysis following dynactin immunodepletion. p150Glued and p50 were immunodepleted from membrane extract of injured and noninjured sciatic nerves; the resulting extract was analyzed by sucrose density sedimentation. The amount of syd, p150Glued, DIC, and KIF5C is constant in extracts prepared from unligated or ligated nerves (Fig. 8 A). In contrast, P-JNK levels are higher in the ligated nerves, as expected. Tubulin and myelin basic protein serve as loading controls. The efficiency of the immunodepletion showed that most of the p150Glued is removed on the antibody-coated beads because none can be detected in the gradient fractions, in contrast to the mock depletion with GFP antibodies (Fig. 8, A and B). No difference was observed in the sedimentation properties of DIC and KIF5C in both ligated and unligated nerves in the presence or absence of the dynactin subunits p50 and p150Glued. DIC peaked at ∼18–19S, whereas KIF5C peaked at ∼4–11S as expected (Kamal et al., 2000). Syd distribution is characterized by two main peaks, one at ∼8S and one at ∼17S, indicating that syd exists at least in two protein complexes of distinct composition. In sciatic nerves injured by ligation, the amount of membrane-associated syd increased in the ∼17S fraction and decreased in the ∼8S fraction compared with the unligated situation (Fig. 8 B, top; and Fig. S2 C). The P-JNK distribution across the sucrose gradients was restricted to the low density fractions (unpublished data; the interaction between P-JNK and dynactin might be weak as reflected by the low amount detected in the dynactin immunoprecipitation). After dynactin depletion, the syd injury-induced increase at ∼17S is lost and a resulting increase at 4–8S is observed. These results suggest that membrane-associated syd exists in several complexes of different sedimentation properties. In uninjured nerves, the syd complex sedimenting at 17S does not reflect dynactin association. However, the increased population of syd at 17S following injury appears to require interaction with the dynactin complex and can account for the observed injury-induced retrograde transport of syd.


Sunday Driver links axonal transport to damage signaling.

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

Syd association with the dynactin complex is increased by injury. The dynactin complex was immunodepleted from membrane-bound material of ligated or unligated sciatic nerves. For dynactin depletion, both p150Glued and p50 antibodies were used, whereas for mock depletion a GFP antibody was used. (A) SDS-PAGE analysis of immunodepletion using dynactin (dyn) or GFP antibodies. Note that input (IN) and immunoprecipitated material (IP) are from equivalent exposures, except for blots with syd and P-JNK antibodies, which required higher exposure times. (B) The unbound (nonimmunoprecipitated) material was sedimented on 5–20% linear sucrose gradients. Fractions were resolved by SDS-PAGE and Western blots were performed using syd, p150Glued, DIC, and KIF5C antibodies. Quantification is expressed as a percentage of total ± SEM (n = 3). Dynactin depletion (closed symbols) and mock GFP depletion (open symbols) is shown. A representative Western blot is shown in the online supplemental material.
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fig8: Syd association with the dynactin complex is increased by injury. The dynactin complex was immunodepleted from membrane-bound material of ligated or unligated sciatic nerves. For dynactin depletion, both p150Glued and p50 antibodies were used, whereas for mock depletion a GFP antibody was used. (A) SDS-PAGE analysis of immunodepletion using dynactin (dyn) or GFP antibodies. Note that input (IN) and immunoprecipitated material (IP) are from equivalent exposures, except for blots with syd and P-JNK antibodies, which required higher exposure times. (B) The unbound (nonimmunoprecipitated) material was sedimented on 5–20% linear sucrose gradients. Fractions were resolved by SDS-PAGE and Western blots were performed using syd, p150Glued, DIC, and KIF5C antibodies. Quantification is expressed as a percentage of total ± SEM (n = 3). Dynactin depletion (closed symbols) and mock GFP depletion (open symbols) is shown. A representative Western blot is shown in the online supplemental material.
Mentions: Although generally increased axonal transport following injury has been demonstrated (Curtis et al., 1998), evidence for injury-induced transport of signaling molecules other than neurotrophins does not exist (Delcroix et al., 2003a). Thus, we tested if sciatic nerve injury results in an increase of syd association with the dynactin complex, which might account for a switch to retrograde transport. By performing sucrose density sedimentation analysis, we observed that the soluble pool of syd is found exclusively at ∼8S (unpublished data), whereas the membrane-associated pool is found in two peaks, ∼8S and ∼17S. To test if the syd peak at ∼17S reports an interaction with dynactin, we performed sucrose density gradient sedimentation analysis following dynactin immunodepletion. p150Glued and p50 were immunodepleted from membrane extract of injured and noninjured sciatic nerves; the resulting extract was analyzed by sucrose density sedimentation. The amount of syd, p150Glued, DIC, and KIF5C is constant in extracts prepared from unligated or ligated nerves (Fig. 8 A). In contrast, P-JNK levels are higher in the ligated nerves, as expected. Tubulin and myelin basic protein serve as loading controls. The efficiency of the immunodepletion showed that most of the p150Glued is removed on the antibody-coated beads because none can be detected in the gradient fractions, in contrast to the mock depletion with GFP antibodies (Fig. 8, A and B). No difference was observed in the sedimentation properties of DIC and KIF5C in both ligated and unligated nerves in the presence or absence of the dynactin subunits p50 and p150Glued. DIC peaked at ∼18–19S, whereas KIF5C peaked at ∼4–11S as expected (Kamal et al., 2000). Syd distribution is characterized by two main peaks, one at ∼8S and one at ∼17S, indicating that syd exists at least in two protein complexes of distinct composition. In sciatic nerves injured by ligation, the amount of membrane-associated syd increased in the ∼17S fraction and decreased in the ∼8S fraction compared with the unligated situation (Fig. 8 B, top; and Fig. S2 C). The P-JNK distribution across the sucrose gradients was restricted to the low density fractions (unpublished data; the interaction between P-JNK and dynactin might be weak as reflected by the low amount detected in the dynactin immunoprecipitation). After dynactin depletion, the syd injury-induced increase at ∼17S is lost and a resulting increase at 4–8S is observed. These results suggest that membrane-associated syd exists in several complexes of different sedimentation properties. In uninjured nerves, the syd complex sedimenting at 17S does not reflect dynactin association. However, the increased population of syd at 17S following injury appears to require interaction with the dynactin complex and can account for the observed injury-induced retrograde transport of syd.

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