Pezo-1 Function in Caenorhabditis elegans

Citation

Brugman, Katherine Irene (2020) Pezo-1 Function in Caenorhabditis elegans. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fz4z-c850. https://resolver.caltech.edu/CaltechTHESIS:05312020-153151158

Abstract

The piezo class of mechanosensative ion channels is a recently discovered class of cation channels with orthologs found in every phylogenetic clade aside from yeast and bacteria. THey are large channels, both in gene length and in overall diameter, with the diameter of the human PIEZO2 protein measuring in at 280 Å in its full homotrimeric form. In addition, like many similar mechanosensitive channels, such as the DEG/ENaC channels, TRP channels, and TREK/TRAAK channels, they have been linked to a number of different functions within drosophila, zebrafish, and mice, including light touch, nociception, blood cell volume regulation, vascular development, and neuropathic pain. Structurally, this channel is intriguing as it possesses no previously categorized structural motifs and is organized into a central pore with a cap, surrounded by three "propeller blade" regions that are theorized to anchor the channel to the membrane and control gating through hydrophobic mismatch based on membrane curvature.

The C. elegans piezo, pezo-1, has not yet been fully characterized, even though a crystal structure of part of this particular piezo was used to assist in the resolution of the first set of cryo-EM images. Here, I generated a number of GFP transcriptional fusions of non-coding potential promoter regions to track the expression of the pezo-1 gene in C. elegans, using these expression patterns to design further experiments. From these expression patterns, I identified expression in a number of neurons of the C. elegans male tail, the primary mating apparatus of the male, and identified these neurons as key neurons as relating to mating. In particular, I identified neurons HOB, PCB, PCC and various ray neurons as potential candidates, which are ciliated neurons theorized to have mechanosensitive properties. In addition, I also identified expression in the vulva muscle and spermatheca of the hermaphrodite, both theorized to be involved in ovulation and egg-laying processes.

From there, I designed CRISPR/Cas9 mutants with defects in pezo-1 in order to investigate the potential link between the pezo-1 expression in those neurons and mating behavior via a mating assay. Similarly, I devised a fecundity assay to investigate the link between pezo-1 expression in ovulation organs and progeny survival. I have discovered that pezo-1 has function in both of these areas, with pezo-1 mutant males demonstrating discrete mating defects that correlate with the expression pattern seen from the GFP transcriptional fusion mutants and with pezo-1 mutant hermaphrodites having much smaller brood sizes than wildtype hermaphrodites. However, I have also discovered that the processes this mechanotransducer is involved in are also more complex than I originally believed, as I discovered that pezo-1 appears to interact with another mechanotransducer, trp-4, illuminating some potentially novel pathway considerations for how these channels overlap in function.

Thesis Files