Understanding the mechanisms of neuronal growth cone guidance and motility is imperative, if we want to develop successful strategies for nerve regeneration after injury and neurodegenerative diseases. Although a large number of axon guidance molecules have been characterized in recent years, there are significant gaps in our understanding of the molecular and cellular mechanisms that the growth cone uses to integrate its sensory, signaling and motile functions. We have recently provided evidence that the immunoglobulin superfamily cell adhesion molecule apCAM mediates growth cone steering by substrate-cytoskeletal coupling. pCAM-actin coupling depends on Src kinase activity and results in actin flow attenuation ollowed by microtubule extension. Recent findings further suggest that microtubules influence Src kinase activity at adhesion sites. The goal of this project is to test the following hypothesis: Src kinase activity and microtubule dynamics regulate apCAM-cytoskeletal coupling in neuronal growth cone steering. Using the well-established high-resolution Aplysia growth cone system, advanced live cell imaging techniques, and a new set of molecular tools for Src tyrosine kinases, we will address three Specific Aims: (1) to determine if microtubules play a role early during apCAM-mediated growth cone steering. We will achieve this goal by combining a novel in vitro growth cone steering assay with microtubule fluorescent speckle microscopy to quantify the dynamic behavior of microtubules early before the major microtubule rearrangement occurs.
The second Aim of this study is: (2) to identify Aplysia Src family kinases, an important group of tyrosine kinases implicated in the regulation of axonal growth, and to determine their subcellular localization, activation state and dynamics in growth cones. To achieve this goal, we will prepare antibodies and EGFP-fusion constructs of newly identified Src kinases in Aplysia.
The third Aim i s: (3) to determine the role of these Src family kinases in apCAM-mediated growth cone steering. Therefore, we will image Src-EGFP protein dynamics during growth cone steering events and test the effect of active and inactive Src mutants on apCAM-actin coupling and growth cone guidance. These studies will not only provide new insights into the role of microtubules and Src kinases in growth cone steering, but also unprecedented information on the dynamic behavior of this key signaling enzyme within a living neuron. Thus, they will have an impact on our understanding of axon guidance and nerve regeneration, as well as of tumor cell metastasis, another motile process, in which Src has been implicated.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Riddle, Robert D
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Purdue University
Schools of Arts and Sciences
West Lafayette
United States
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He, Yingpei; Ren, Yuan; Wu, Bingbing et al. (2015) Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones. Mol Biol Cell 26:3229-44
Munnamalai, Vidhya; Weaver, Cory J; Weisheit, Corinne E et al. (2014) Bidirectional interactions between NOX2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones. J Neurochem 130:526-40
Martines, E; Zhong, J; Muzard, J et al. (2012) Single-molecule force spectroscopy of the Aplysia cell adhesion molecule reveals two homophilic bonds. Biophys J 103:649-57
Kilinc, Devrim; Blasiak, Agata; O'Mahony, James J et al. (2012) Magnetic tweezers-based force clamp reveals mechanically distinct apCAM domain interactions. Biophys J 103:1120-9
Suter, Daniel M; Hollenbeck, Peter J (2011) How to get on the right track. Nat Neurosci 15:7-8
Suter, Daniel M (2011) Live cell imaging of neuronal growth cone motility and guidance in vitro. Methods Mol Biol 769:65-86
Suter, Daniel M; Miller, Kyle E (2011) The emerging role of forces in axonal elongation. Prog Neurobiol 94:91-101
Decourt, Boris; Munnamalai, Vidhya; Lee, Aih Cheun et al. (2009) Cortactin colocalizes with filopodial actin and accumulates at IgCAM adhesion sites in Aplysia growth cones. J Neurosci Res 87:1057-68
Munnamalai, Vidhya; Suter, Daniel M (2009) Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth. J Neurochem 108:644-61
Xiong, Ying; Lee, Aih Cheun; Suter, Daniel M et al. (2009) Topography and nanomechanics of live neuronal growth cones analyzed by atomic force microscopy. Biophys J 96:5060-72

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