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MBC in Press, published online ahead of print August 20, 2008
Mol. Biol. Cell 10.1091/mbc.E08-06-0603

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Submitted on June 13, 2008
Revised on July 30, 2008
Accepted on August 13, 2008

Microtubule-mediated Src Tyrosine Kinase Trafficking in Neuronal Growth Cones

Bingbing Wu,* Boris Decourt,* Muhammad A. Zabidi,* Levi T. Wuethrich,* William H. Kim,* Zhigang Zhou,{dagger} Keira MacIsaac,* and Daniel M. Suter{ddagger}

*Department of Biological Sciences, {ddagger}Bindley Bioscience Center, and {dagger}Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907

Monitoring Editor: Paul Forscher

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone and have been implicated in axon guidance; however, the detailed localization, trafficking and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-EGFP-positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones implicating Src2 in controlling the size of filopodia and lamellipodia.

Address correspondence to: Daniel M. Suter (dsuter{at}purdue.edu)




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