91Ô­´´

Analysis of animals expressing both tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, a known cargo in EVs released from the same ciliated sensory neurons, showed that the two fluorescent proteins do not colocalize in EVs (Fig. 2). Our goal is to use strengths of theÌýC. elegansÌýsystem to define EV biogenesis mechanisms and understand how EV cargo sorting specificity is achieved. Ìý

Identification of CALHM channel regulatorsÌýWe are also utilizing the powerfulÌýC. elegansÌýmodel to gain critical insights into cellular CALHM channel function. Our goal is to identify and characterize CLHM-1 regulators in order to determine the molecular mechanisms that control channel localization and function. We have isolated CLHM-1 regulators inÌýC. elegansÌýby performing an unbiased forward genetic screen for suppressors of toxicity associated with CLHM-1 over-expression. After identifying the causative mutations by a whole genome sequencing strategy, we will characterize the mutants using genetic, behavioral and cell biological approaches.

Impact of Diet on Amyloid-beta Toxicity inÌýC. elegansÌýWe use aÌýC. elegansÌýmodel of Alzheimer’s disease (AD) to identify factors that impact Aβ toxicity, a causative factor in AD pathogenesis. Expression of human Aβ1-42 inÌýC. elegansÌýcauses fully penetrant, age-dependent paralysis. We found that loss of CLHM-1 had no effect on Aβ toxicity, however, while conducting these experiments we discovered that the type ofÌýE. coliÌýdiet that Aβ-expressing animals consume alters paralysis rate. Our goal is to determine how diet affects Aβ levels, mitochondrial morphology, mitochondrial function, and gene expression in Aβ-expressingÌýC. elegans.

Characterization of novel regulators of post-synaptic signaling at the NMJÌýC. elegansÌýbody-wall muscles are comparable to vertebrate skeletal muscles and provide an excellent model to study neuromuscular transmission. To identify novel factors that regulate post-synaptic cholinergic signaling we carried out a genome wide RNAi screen inÌýC. elegansÌýfor gene knockdowns that cause hypersensitivity or resistance to the AChR agonist levamisole. We discovered 156 gene knockdowns that caused altered levamisole response. Our goal is to define the mechanism(s) by which neuromuscular transmission is altered by determining how the gene knockdowns affect locomotion, signaling, and synaptic structure using biomechanical, optogenetic and imaging approaches.Ìý

A rapid, super-selective method for detection of single nucleotide variantsÌýWith wide spread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies there is pressing need for an efficient and low-cost strategy to detect single nucleotide variants. We developed a rapid and inexpensive method for detection of single nucleotide variants by adapting superselective primers for end-point PCR. Each superselective primer contains an anchor, bridge, and foot with the last nucleotide in the foot region determining specificity for the mutant allele versus wild type (Fig. 3). We explored how length, stability and sequence composition of each segment affected primer selectivity and developed simple rules for primer design (manuscript in preparation). We have demonstrated the utility of superselective primers for routine genotyping, detection of genome editing events, and colony PCR to identify successful site-directed mutagenesis constructs. Additional ongoing projects in the lab are aimed at developing novel genetics methods.

Previous Group Members

Kirsten KervinÌýGraduate Student (BS Delaware State University, MS 91Ô­´´) Impact of diet on amyloid-beta toxicity; NMJ signaling. Current - Laboratory Technician II at WuXi AppTec.

Elaine MillerÌýResearch Associate (BS, University of California – Davis) CLHM-1 regulators; NMJ signaling. Current – graduate student at George Washington University.

Shrey PatelÌýUndergraduate Biological Sciences Major (BA, 91Ô­´´) NMJ signaling. Current – medical student at Drexel University College of Medicine.