The long term objective of this proposal is to understand the molecular mechanisms of growth cone motility in developing neurons. Axons are guided by extracellular cues that direct filopodial motility by locally affecting cytoskeletal dynamics. Understanding the molecular basis of this is required to understand how neurocircuitry is formed during embryonic development and to establish what factors can cause neurological birth defects. Moreover, these molecular mechanisms are likely to be used in nerve regeneration and an understanding of them will aid in designing treatment after nerve injury. Considerable work in neural development, cell biology, and signal transduction have identified candidate proteins that may be involved in filopodial motility and guidance but establishing their in vivo function in neuronal growth cones has been difficult. The long term objective will be addressed by applying microscale chromophore assisted laser inactivation (micro-CALI), a method developed in this laboratory, to inactivate specific intracellular proteins with an unprecedented level of spatial and temporal resolution. CALI has been rigorously tested and used to determine the in vivo roles of membrane proteins in neural development. It is timely to use this technique to address the molecular mechanisms of growth cone motility. Specificically, the in vivo roles of talin, vinculin, pp60c-src, and the myosins in filopodial motility will be determined by focally inactivating them in growth cones and observing the resulting behavior by video- enhanced microscopy and quantitative morphometry. Experiments are also proposed to ask if these proteins play a role in substrate-mediated guidance by using micro- CALI as growth cones reach borders on patterned substrates. These studies will be done using chick dorsal root ganglion neurons in culture, a well characterized and manipulatable system for which there exist antibodies against many proteins that are potentially involved in growth cone motility. The proposed experiments are focused on these proteins because our preliminary experiments coupled with in vitro biochemical data suggest a model for how they function and interact in the extension and regulation of filopodia. Micro-CALI will be applied to test this model by inactivating these proteins in combination to give specific phenotypes that will support or refute the proposed interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS034699-08
Application #
6539846
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Mamounas, Laura
Project Start
1999-02-01
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
8
Fiscal Year
2002
Total Cost
$330,693
Indirect Cost
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Yimlamai, Dean; Konnikova, Liza; Moss, Larry G et al. (2005) The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis. Dev Biol 279:44-57
Wong, Eric V; Kerner, Julie A; Jay, Daniel G (2004) Convergent and divergent signaling mechanisms of growth cone collapse by ephrinA5 and slit2. J Neurobiol 59:66-81
Eustace, Brenda K; Jay, Daniel G (2003) Fluorophore-assisted light inactivation for multiplex analysis of protein function in cellular processes. Methods Enzymol 360:649-60
Hauptschein, Robert S; Eustace, Brenda K; Jay, Daniel G (2002) Global high-throughput screens for cellular function. Exp Hematol 30:381-7
Sakurai, Takashi; Wong, Eric; Drescher, Uwe et al. (2002) Ephrin-A5 restricts topographically specific arborization in the chick retinotectal projection in vivo. Proc Natl Acad Sci U S A 99:10795-800
Diefenbach, Thomas J; Latham, Vaughan M; Yimlamai, Dean et al. (2002) Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone. J Cell Biol 158:1207-17
Jay, D G (2001) A Src-astic response to mounting tension. J Cell Biol 155:327-30
Buchstaller, A; Jay, D G (2000) Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins. Microsc Res Tech 48:97-106
Liu, C W; Lee, G; Jay, D G (1999) Tau is required for neurite outgrowth and growth cone motility of chick sensory neurons. Cell Motil Cytoskeleton 43:232-42
Castelo, L; Jay, D G (1999) Radixin is involved in lamellipodial stability during nerve growth cone motility. Mol Biol Cell 10:1511-20

Showing the most recent 10 out of 11 publications