Determining The Impact Of Calcium-Atpase Activity In Hyperstimulated C. Elegans Muscles
Date
2022-05
Authors
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Publisher
University of Delaware
Abstract
Calcium-ATPases are vital pumps in the plasma membrane and sarcoplasmic
reticulum that are required for removal of calcium (Ca2+) against the concentration
gradient to maintain low intracellular Ca2+. At the neuromuscular junction (NMJ),
binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic
receptors leads to an increase in intracellular Ca2+ and muscle contraction.
Reducing intracellular Ca2+ after muscle contraction is required to enable relaxation.
As in vertebrate skeletal muscle, activation of ACh receptors (AChRs) on the body
wall muscles of the model organism C. elegans cause muscle contraction. A genome
wide RNAi screen for altered sensitivity to the AChR agonist levamisole showed that
loss of MCA-3, a plasma membrane Ca2+-ATPase (PMCA), resulted in a levamisole resistant phenotype. This suggests that another mechanism is more efficient in
reducing intracellular Ca2+ when MCA-3 is not present. SCA-1 is a sarcoplasmic
reticulum Ca2+-ATPase (SERCA) in C. elegans body wall muscles. SERCAs move
two Ca2+ for every molecule of ATP, while PMCAs are less efficient and move one
Ca2+ for every molecule of ATP. When cellular ATP is low, Ca2+ cannot be pumped
out of the cytoplasm and the muscles remain contracted. I hypothesize that mutation of
each gene individually will cause altered phenotype and paralysis rates due to the
difference in ATP utilization. I performed behavioral assays to determine levamisole
induced phenotypes. sca-1 mutation and RNAi knockdown resulted in a significant
levamisole hypersensitive phenotype.
Next, I looked to investigate the molecular causes of the observed paralysis.
ATP levels were quantified in RNAi knockdown animals after 0, 30, and 60 minutes
of levamisole treatment. sca-1 knockdown depleted ATP significantly over the first 30
minutes, while ATP depletion in the mca-3 knockdown did not occur until the last 30
minutes of levamisole treatment. Finally, I conducted imaging tracking intracellular
Ca2+ using LSM880 confocal microscopy. I optimized imaging methods and
parameters that show promise for future investigation of mutants and RNAi
knockdowns. In conclusion, SCA-1 plays an important role in reducing intracellular
Ca2+, and ATP availability plays a role in levamisole paralysis rate.
Description
Keywords
Calcium, C.elegans, Levamisole