Biology & Sustainability
The Development of a Novel Fluorophore-Based Method to Investigate Regulatory Neuronal Proteins in Caenorhabditis elegans
Document Type
Poster Presentation
Location
Indianapolis, IN
Subject Area
Biology & Sustainability
Start Date
11-4-2014 8:30 AM
End Date
11-4-2014 9:30 AM
Sponsor
Jeremy Johnson (Butler University), Jennifer Kowalski (Butler University)
Description
Neurons process, store, and transmit information throughout the nervous system, and their function is regulated by hundreds of proteins. Malfunctioning of these proteins can result in neurological and neurodegenerative disorders. An excellent model organism in which to investigate regulatory neuronal proteins and their mechanisms of action is the roundworm Caenorhabditis elegans, as it is amenable to genetic manipulations and shares conservation of neuronal proteins and nervous system structure with humans. Traditionally, proteins of interest have been studied in vivo by fluorescent tagging. With one fluorescent tag per protein, however, this method cannot quantify subtle changes in protein levels, such as those due to the action of regulatory mechanisms. Our goal is to develop a more sensitive in vivo method to amplify the fluorescent signal and allow for rapid quantification of changes in neuronal protein abundance. To achieve this, we plan to deliver a fluorophore masked by an ester chemical group to C. elegans expressing a neuronal protein tagged with porcine liver esterase (PLE), an enzyme able to activate the masked fluorophores. Since fluorophore delivery is hindered by the cuticle, a proteinaceous covering around the worms, we synthesized two ester-masked fluorescein derivatives with hydrophobic tails to aid uptake into the plasma membranes of externally exposed sensory neurons. As proof of principle, we are constructingtransgenic C. elegansstrains with neuronal expression of PLE and testing for neuron-specific activation of our ester-masked fluorophores. Our long-term goals entail using this approach to screen for novel genes involved in regulating neuronal function.
The Development of a Novel Fluorophore-Based Method to Investigate Regulatory Neuronal Proteins in Caenorhabditis elegans
Indianapolis, IN
Neurons process, store, and transmit information throughout the nervous system, and their function is regulated by hundreds of proteins. Malfunctioning of these proteins can result in neurological and neurodegenerative disorders. An excellent model organism in which to investigate regulatory neuronal proteins and their mechanisms of action is the roundworm Caenorhabditis elegans, as it is amenable to genetic manipulations and shares conservation of neuronal proteins and nervous system structure with humans. Traditionally, proteins of interest have been studied in vivo by fluorescent tagging. With one fluorescent tag per protein, however, this method cannot quantify subtle changes in protein levels, such as those due to the action of regulatory mechanisms. Our goal is to develop a more sensitive in vivo method to amplify the fluorescent signal and allow for rapid quantification of changes in neuronal protein abundance. To achieve this, we plan to deliver a fluorophore masked by an ester chemical group to C. elegans expressing a neuronal protein tagged with porcine liver esterase (PLE), an enzyme able to activate the masked fluorophores. Since fluorophore delivery is hindered by the cuticle, a proteinaceous covering around the worms, we synthesized two ester-masked fluorescein derivatives with hydrophobic tails to aid uptake into the plasma membranes of externally exposed sensory neurons. As proof of principle, we are constructingtransgenic C. elegansstrains with neuronal expression of PLE and testing for neuron-specific activation of our ester-masked fluorophores. Our long-term goals entail using this approach to screen for novel genes involved in regulating neuronal function.