This proposal concerns the molecular and cellular mechanisms that determine synaptic connectivity in developing nervous systems. It focuses on a set of Drosophila neuronal cell surface/signal transduction proteins that we have been studying for many years: the transmembrane receptor tyrosine phosphatases (RPTPs). The six Drosophila RPTPs are central regulators of axon guidance and synaptogenesis. They have been extensively investigated using genetics, but we still known relatively little about the signaling pathways in which they function. This proposal is directed toward identification and characterization of components of these pathways.
Specific aims 1 and 2 concern new methods for identification of RPTP ligands and coreceptors. We have already defined a Ptp10D binding protein, Sas, using one of these methods, and will characterize Sas's roles in regulation of Ptp10D function in vivo. We have also shown that a signal from glia to neurons is required for axonal expression of a Ptp99A binding protein. We will follow up on these discoveries, and also continue our search for new binding proteins for four different RPTPs.
Specific aim 3 describes genetic screens for components of RPTP signaling pathways. We recently defined a remarkable and unique protein trafficking phenotype in embryos lacking both Type III RPTPs, Ptp10D and Ptp4E. Tracheal cells need to make new apical membrane in order to create the lumens of tracheal tubes. In the double mutant embryos, apical proteins accumulate in large intracellular vacuoles ("bubbles"). Basolateral proteins are localized normally. Our results show that EGFR, Rho family GTPases, and Rab GTPases are involved in generation of this phenotype. We suggest that it occurs because EGFR and Rho activites are upregulated when the RPTPs are absent. This shifts the balance between endocytosis and exocytosis, favoring accumulation of apical proteins in fused endocytic vesicles at the expense of the luminal surface. We will examine whether some of these pathway components are also used for regulation of axon guidance by these RPTPs. We will use the tracheal phenotype as the basis for F1 and F2 genetic screens to find new proteins in the RPTP signaling pathways, and will evaluate the functions of the genes we discover in both neurons and tracheae.
Specific aim 4 concerns a new method for identification of in vivo substrates of RPTPs. We will express "substrate trap" mutants of RPTPs in neurons and tracheae, purify tyrosine-phosphorylated proteins that associate with the traps, and identify these proteins by mass spectrometry. We will then characterize the roles of these putative substrates in the RPTP pathways using genetics, and reconstruct their phosphorylation and dephosphorylation in cell culture.

Public Health Relevance

to human health: This is a basic research project to discover mechanisms involved in creation of neuronal circuits during development. Although the work is conducted in Drosophila, most of the genes we are studying have human counterparts. We hope to reveal general principles that will facilitate an understanding of how human brain wiring is controlled before and after birth. Knowledge about wiring mechanisms may help researchers to understand diseases in which neuronal connectivity patterns are altered. These include schizophrenia and autism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS028182-24
Application #
8399723
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Riddle, Robert D
Project Start
1990-01-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
24
Fiscal Year
2013
Total Cost
$325,047
Indirect Cost
$118,175
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Lee, Hyung-Kook Peter; Cording, Amy; Vielmetter, Jost et al. (2013) Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication. Neuron 78:813-26
Menon, Kaushiki P; Carrillo, Robert A; Zinn, Kai (2013) Development and plasticity of the Drosophila larval neuromuscular junction. Wiley Interdiscip Rev Dev Biol 2:647-70
Ozkan, Engin; Carrillo, Robert A; Eastman, Catharine L et al. (2013) An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks. Cell 154:228-39
Al-Anzi, Bader; Armand, Elena; Nagamei, Paul et al. (2010) The leucokinin pathway and its neurons regulate meal size in Drosophila. Curr Biol 20:969-78
Wright, Ashley P; Fox, A Nicole; Johnson, Karl G et al. (2010) Systematic screening of Drosophila deficiency mutations for embryonic phenotypes and orphan receptor ligands. PLoS One 5:e12288
Salazar, Anna M; Silverman, Edward J; Menon, Kaushiki P et al. (2010) Regulation of synaptic Pumilio function by an aggregation-prone domain. J Neurosci 30:515-22
Bugga, Lakshmi; Ratnaparkhi, Anuradha; Zinn, Kai (2009) The cell surface receptor Tartan is a potential in vivo substrate for the receptor tyrosine phosphatase Ptp52F. Mol Cell Biol 29:3390-400
Lee, Hyung-Kook Peter; Wright, Ashley P; Zinn, Kai (2009) Live dissection of Drosophila embryos: streamlined methods for screening mutant collections by antibody staining. J Vis Exp :
Lim, Angeline; Kraut, Rachel (2009) The Drosophila BEACH family protein, blue cheese, links lysosomal axon transport with motor neuron degeneration. J Neurosci 29:951-63
Jeon, Mili; Zinn, Kai (2009) Receptor tyrosine phosphatases control tracheal tube geometries through negative regulation of Egfr signaling. Development 136:3121-9

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