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Originally published as MBC in Press, 10.1091/mbc.E08-06-0603 on August 20, 2008

Vol. 19, Issue 11, 4611-4627, November 2008

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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

Submitted June 13, 2008; Revised July 30, 2008; Accepted August 13, 2008
Monitoring Editor: Paul Forscher

InCytes from MBC

Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they 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 we 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-enhanced green fluorescent protein–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.

This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-06-0603) on August 20, 2008.

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

Abbreviations used: ASW, artificial seawater; C, central; CSB, cytoskeleton stabilizing buffer; DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; FRAP, fluorescent recovery after photobleaching; FSM, fluorescent speckle microscopy; MT, microtubule; P, peripheral; PTK, protein tyrosine kinase; RACE, rapid amplification of cDNA ends; SH, Src homology; SYF, cell line from Src/Yes/Fyn triple knockout mouse; T, transition.


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