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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 is a peripheral membrane protein associated with axonal vesicular structures in mouse sciatic nerve. (A) After flotation on sucrose step gradients, syd, KIF5C, JNK3, and P-JNK are present in both the soluble fraction (40% sucrose) and the membrane fraction (35/25 interface). The 40/35 interface contains both soluble and membrane-bound proteins (note that JNK antibodies recognize the 46- and 54-kd isoforms). (B) Sciatic nerve PNS or floating membrane fractions (memb) were treated with Triton X-114 at 4°C. Syd and JNK3 are found in the aqueous phase (note that the top band recognizing the JNK3 54-kd isoform is below detection level at this exposure time in the membrane extraction). (C) After carbonate wash of sciatic nerve PNS, a significant amount of syd remains associated with membranes. (D) Brain membrane fractions were treated with trypsin (T) in the presence or absence of soybean trypsin inhibitor (Ti) or Triton X-100 (det), as indicated. APP is protected from trypsin digestion, whereas syd is not detected with NH2- or COOH-terminal antibodies after digestion. (E) Sciatic nerve cross sections were stained for syd (anti-JIP3) and S100, a Schwann cell marker. Deconvolution analysis shows punctate syd staining. Red arrows, syd puncta; green arrows, Schwann cells. (F) Sciatic nerve cross sections were stained for syd (syd N-ter) and COX1. No colocalization is observed. Red arrows, syd puncta; green arrows, COX1 puncta; arrowheads, myelin outer layer. Bars, 5 μm.
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fig1: Syd is a peripheral membrane protein associated with axonal vesicular structures in mouse sciatic nerve. (A) After flotation on sucrose step gradients, syd, KIF5C, JNK3, and P-JNK are present in both the soluble fraction (40% sucrose) and the membrane fraction (35/25 interface). The 40/35 interface contains both soluble and membrane-bound proteins (note that JNK antibodies recognize the 46- and 54-kd isoforms). (B) Sciatic nerve PNS or floating membrane fractions (memb) were treated with Triton X-114 at 4°C. Syd and JNK3 are found in the aqueous phase (note that the top band recognizing the JNK3 54-kd isoform is below detection level at this exposure time in the membrane extraction). (C) After carbonate wash of sciatic nerve PNS, a significant amount of syd remains associated with membranes. (D) Brain membrane fractions were treated with trypsin (T) in the presence or absence of soybean trypsin inhibitor (Ti) or Triton X-100 (det), as indicated. APP is protected from trypsin digestion, whereas syd is not detected with NH2- or COOH-terminal antibodies after digestion. (E) Sciatic nerve cross sections were stained for syd (anti-JIP3) and S100, a Schwann cell marker. Deconvolution analysis shows punctate syd staining. Red arrows, syd puncta; green arrows, Schwann cells. (F) Sciatic nerve cross sections were stained for syd (syd N-ter) and COX1. No colocalization is observed. Red arrows, syd puncta; green arrows, COX1 puncta; arrowheads, myelin outer layer. Bars, 5 μm.

Mentions: Initial data and motif analysis suggested that syd might be a transmembrane protein (Bowman et al., 2000). To test this hypothesis, we performed a detailed biochemical characterization. In sucrose step flotation gradients, in which sciatic nerve extract was bottom loaded, syd, together with the motor protein kinesin-I (KIF5C), was present in both the soluble fraction (40% sucrose) and the different sucrose interfaces that are characteristic of membrane compartments (Fig. 1 A). The transmembrane protein APP is found exclusively in the 35/25 floating fractions, whereas myelin basic protein is found exclusively in the 25/8 interface. Syd is a scaffolding protein for the JNK group of stress-activated protein kinases and shows preferential binding of the neuronal enriched isoform JNK3 over the ubiquitous JNK1 and JNK2 (Ito et al., 1999; Kelkar et al., 2000). Thus, we examined the distribution of JNK3 in the sucrose step gradient. We find a small fraction of JNK3 associated with membrane in the floating fractions. The activated form of JNK, detected with an antibody against the phosphorylated form (P-JNK), is also present in the 35/25 interface.


Sunday Driver links axonal transport to damage signaling.

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

Syd is a peripheral membrane protein associated with axonal vesicular structures in mouse sciatic nerve. (A) After flotation on sucrose step gradients, syd, KIF5C, JNK3, and P-JNK are present in both the soluble fraction (40% sucrose) and the membrane fraction (35/25 interface). The 40/35 interface contains both soluble and membrane-bound proteins (note that JNK antibodies recognize the 46- and 54-kd isoforms). (B) Sciatic nerve PNS or floating membrane fractions (memb) were treated with Triton X-114 at 4°C. Syd and JNK3 are found in the aqueous phase (note that the top band recognizing the JNK3 54-kd isoform is below detection level at this exposure time in the membrane extraction). (C) After carbonate wash of sciatic nerve PNS, a significant amount of syd remains associated with membranes. (D) Brain membrane fractions were treated with trypsin (T) in the presence or absence of soybean trypsin inhibitor (Ti) or Triton X-100 (det), as indicated. APP is protected from trypsin digestion, whereas syd is not detected with NH2- or COOH-terminal antibodies after digestion. (E) Sciatic nerve cross sections were stained for syd (anti-JIP3) and S100, a Schwann cell marker. Deconvolution analysis shows punctate syd staining. Red arrows, syd puncta; green arrows, Schwann cells. (F) Sciatic nerve cross sections were stained for syd (syd N-ter) and COX1. No colocalization is observed. Red arrows, syd puncta; green arrows, COX1 puncta; arrowheads, myelin outer layer. Bars, 5 μm.
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Related In: Results  -  Collection

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fig1: Syd is a peripheral membrane protein associated with axonal vesicular structures in mouse sciatic nerve. (A) After flotation on sucrose step gradients, syd, KIF5C, JNK3, and P-JNK are present in both the soluble fraction (40% sucrose) and the membrane fraction (35/25 interface). The 40/35 interface contains both soluble and membrane-bound proteins (note that JNK antibodies recognize the 46- and 54-kd isoforms). (B) Sciatic nerve PNS or floating membrane fractions (memb) were treated with Triton X-114 at 4°C. Syd and JNK3 are found in the aqueous phase (note that the top band recognizing the JNK3 54-kd isoform is below detection level at this exposure time in the membrane extraction). (C) After carbonate wash of sciatic nerve PNS, a significant amount of syd remains associated with membranes. (D) Brain membrane fractions were treated with trypsin (T) in the presence or absence of soybean trypsin inhibitor (Ti) or Triton X-100 (det), as indicated. APP is protected from trypsin digestion, whereas syd is not detected with NH2- or COOH-terminal antibodies after digestion. (E) Sciatic nerve cross sections were stained for syd (anti-JIP3) and S100, a Schwann cell marker. Deconvolution analysis shows punctate syd staining. Red arrows, syd puncta; green arrows, Schwann cells. (F) Sciatic nerve cross sections were stained for syd (syd N-ter) and COX1. No colocalization is observed. Red arrows, syd puncta; green arrows, COX1 puncta; arrowheads, myelin outer layer. Bars, 5 μm.
Mentions: Initial data and motif analysis suggested that syd might be a transmembrane protein (Bowman et al., 2000). To test this hypothesis, we performed a detailed biochemical characterization. In sucrose step flotation gradients, in which sciatic nerve extract was bottom loaded, syd, together with the motor protein kinesin-I (KIF5C), was present in both the soluble fraction (40% sucrose) and the different sucrose interfaces that are characteristic of membrane compartments (Fig. 1 A). The transmembrane protein APP is found exclusively in the 35/25 floating fractions, whereas myelin basic protein is found exclusively in the 25/8 interface. Syd is a scaffolding protein for the JNK group of stress-activated protein kinases and shows preferential binding of the neuronal enriched isoform JNK3 over the ubiquitous JNK1 and JNK2 (Ito et al., 1999; Kelkar et al., 2000). Thus, we examined the distribution of JNK3 in the sucrose step gradient. We find a small fraction of JNK3 associated with membrane in the floating fractions. The activated form of JNK, detected with an antibody against the phosphorylated form (P-JNK), is also present in the 35/25 interface.

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