The guidance of growing neurons in the developing embryo is an essential step in the establishment of neuronal connections, a requirement for nervous system function. Although a large number of guidance molecules and their neuronal receptors have been identified, little is known at the cellular level about how receptor activation gives rise to the changes in cell shape that are required for guided axon growth. The study of adhesion receptors in neurons and other migrating cells has suggested that the generation of cellular traction forces through sites of cell contact with the environment may play a central role in the regulation of cell translocation. The overall aim of this proposal is to define the mechanism underlying the generation of traction forces in the growing neuron, focusing on the neuronal immunoglobulin (Ig) protein L1 as a model adhesion receptor. Our preliminary data suggest that L1 family members interact with force-generating components of the cytoskeleton. Based on this observation, we propose three major aims: 1) To characterize the regulation and structural requirements of L1-cytoskeleton interactions and the resulting cellular traction forces. 2) To quantify the kinetics of L1 phosphorylation at a site in the cytoplasmic tail known to regulate the binding of the cytoskeleton linker protein ankyrinB. 3) To use in vitro neurite outgrowth assays on substrates coated with L1 ligands to determine how domains identified as being essential to L1 function in 1 and 2 above are required for L1-mediated axon growth. Using a combination of high-resolution video microscopy and an optical gradient laser trap (laser tweezers) to pull on microscopic beads bound to cell-surface L1, we can monitor the regulation of L1 function. These approaches permit the detection of L1-cytosksleton interactions with high spatial and temporal accuracy, allowing us to examine directly how L1 function is modulated by ligand activation, crosslinking and phosphorylation. Mutations in the gene encoding L1 in humans leads to a complex of neurological disorders including spastic paraplegia and mental retardation, suggesting that L1 plays an important role in neural development. By characterizing L1 function at a cellular and biophysical level, we can begin to develop a detailed understanding of the mechanisms underlying directed neuronal growth. Moreover, the information that we gain from studying adhesion receptors in neurons is likely to inform our understanding of cell migration in a variety of other systems, ranging from immune system function to tumor cell metastasis.
| Sakurai, Takeshi; Gil, Orlando D; Whittard, John D et al. (2008) Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylation. J Neurosci Res 86:2602-14 |
| Whittard, John D; Sakurai, Takeshi; Cassella, Melanie R et al. (2006) MAP kinase pathway-dependent phosphorylation of the L1-CAM ankyrin binding site regulates neuronal growth. Mol Biol Cell 17:2696-706 |
| Gil, Orlando D; Sakurai, Takeshi; Bradley, Ann E et al. (2003) Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface. J Cell Biol 162:719-30 |
