Localization of pezo-1 gene expression is responsible for transitioning meiotic to mitotic replication in oocytes of Caenorhabditis elegans Restricted; Files Only

Noetzel, Courtney (Spring 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/w0892c067?locale=en
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Abstract

Fertilization in the nematode Caenorhabditis elegans is extraordinarily efficient so that every sperm fertilizes an oocyte entering the wild type hermaphrodite fertilization chamber, the spermatheca. These fertilized oocytes exit the spermatheca, enter the uterus, and transition from meiosis to mitosis as embryogenesis begins. C. elegans mutants that affect sperm function, such as fer-1, produce defective sperm that are unable to fertilize oocytes. The unfertilized oocytes exit meiosis and become endomitotic because repeated rounds of DNA synthesis occur in the absence of cell division, creating an “Emo” phenotype. A mutant phenotype exists where unfertilized oocytes remain fixed in prophase I of meiosis, creating a “nonEmo” phenotype. The gene responsible for this transition from meiosis to mitosis was identified as pezo-1. Tagging the native pezo-1 gene using CRISPR­ with mCherry revealed widespread expression of PEZO-1::mCherry including in both the spermatheca and oocytes. Analyzing loss of function mutants revealed which of the 12 alternative splice forms of the wild type pezo-1 gene are required for wild type (Emo) PEZO-1 function, allowing CRISPR genome engineering to be used to eliminate many irrelevant isoforms. Isoform b, which produces the smallest protein with wild type function, encodes a 2,400 amino acid, predicted to be a 29-pass transmembrane protein that functions as a mechanosensitive ion channel. Looking at PEZO-1::mCherry expression of PEZO-1 isoforms reveals that itretains function and expression across the various isoforms despite large differences in the exons and large intron present. Furthermore, using a chemical agonist that constitutively opens the PEZO-1 ion channel is associated with the nonEmo phenotype. Lastly, using an auxin inducible degradation (AID) system and a GFP targeting nanobody, PEZO-1 is selectively degraded in either the germline or spermatheca, and the associated nonEmo phenotype implies PEZO-1 needs to be expressed in the germline or likely the spermatheca to cause the Emo phenotype. Together, these data give an understanding of how pezo-1 expression across the isoforms, conformation of the channel, and tissue localization of the protein cause the Emo phenotype. With this understanding, the mechanism of how pezo-1 expression in the plasma membrane of oocytes causes the Emo phenotype and why the protein evolved may be discovered.

Table of Contents

Chapter 1: Introduction (1)

Chapter 2: Analysis of PEZO-1 Isoforms Using Fluorescence Microscopy

A. Introduction (4)

B. Methods (5)

C. Materials (6)

D. Results (6)

Chapter 3: Using a PEZO-1 Agonist to Infer the Conformation of PEZO-1 that is Needed to Cause the Emo Phenotype

A. Introduction (8)

B. Methods (8)

C. Results (9)

D. Discussion (9)

Chapter 4: Using Auxin Inducible Degradation (AID) System to Infer Tissue Expression of PEZO-1 Required to Produce the Emo Phenotype

A. Introduction (11)

B. Methods (12)

C. Results (17)

D. Discussion (19)

Chapter 5: Using a GFP Targeting Nanobody to Infer Tissue Specific Expression of PEZO-1 Required to Produce the Emo Phenotype

A. Introduction (21)

B. Methods (21)

C. Results (23)

D. Discussion (23)

Chapter 6: Conclusions and Future Directions (25)

Figures and Tables (28)

References (42)

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