LOCALIZATION AND TRAFFICKING OF THE CAENORHABDITIS ELEGANS DOPAMINE TRANSPORTER (DAT-1).
PAUL W. MCDONALD
Dissertation under the direction of Professor Randy D. Blakely, Ph.D.
Reuptake of dopamine (DA) through the dopamine transporter (DAT) is the primary mechanism by which DA signaling is terminated at the synapse. Drugs of abuse such as cocaine and amphetamine target DAT, altering DA signaling in the central nervous system. Mechanisms that localize DAT to synapses and control its activity remain ill defined. The soil nematode C. elegans, elaborates a cocaine-sensitive DAT (DAT-1), which is expressed in all the DA neurons of the worm and offers advantages over studies of DATs performed in mammalian systems.
To advance studies of DAT-1 localization and trafficking mechanisms within an intact nervous system in vivo, I first generated DAT-1 specific antibodies to establish wild type DAT-1 localization. Once established, I expressed green fluorescent protein (GFP) fused to NH2-terminal of DAT-1 (GFP:DAT-1) in the DA neurons and examined localization in different compartments within a single neuron. Consistent with native antibody studies, GFP:DAT-1 is expressed throughout the DA neuron, with a synaptic accumulation of signal not seen with cytosolic GFP. Biophysical studies using fluorescent recovery after photobleaching (FRAP) reveal that this accumulation is predominantly immobile, whereas the cell body GFP:DAT-1 remains free to diffuse along the plasma membrane of the neuron.
Trafficking studies examining GFP:DAT-1 localization using known synaptic determinant mutants and mutants generated in a forward genetic screen reveal that specific residues within DAT-1 are important for export to both dendritic and synaptic areas, whereas correct localization of DAT-1 is independent of the kinesin motor protein UNC-104. Examination of a truncated version of the GFP:DAT-1 fusion that lacks specific sequence implicated in both protein-protein interactions (GFP:DAT-1(delta IML)) reveal that expression of this mutant transporter in vivo results in protein instability and cell body accumulation, with little effect on synaptic targeting.
Taken together, theses studies develop both a novel system for examination of fusion protein localization and function in vivo and they more specifically report, for the first time, the localization of a cocaine-sensitive DAT in intact DA neurons in vivo setting the stage for future targeted studies of DAT-1.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03292006-174955 |
| Date | 07 April 2006 |
| Creators | McDonald, Paul William |
| Contributors | Lou Defelice, Randy Blakely, Al George Jr., Andy Link, David Miller |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03292006-174955/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0014 seconds