Submission Date


Document Type

Paper- Restricted to Campus Access




Rebecca Lyczak

Committee Member

Dale Cameron

Committee Member

Lisa Grossbauer

Department Chair

Rebecca Lyczak

Project Description

PAM-1 is a puromycin sensitive aminopeptidase responsible for cleaving amino acids from the N-terminus of unknown target proteins. In our model system, a microscopic nematode known as C. elegans, functional PAM-1 is required for proper polarity establishment, centrosome positioning, asymmetric division, cytoskeleton development, as well as meiosis (Lyczak, et al., 2006). Due to these defects, pam-1 mutants have a reduced hatch rate to around 12%, compared to the wildtype hatch rate of 95%. A secondary suppressor of pam-1, a mutant gene for the inhibitory kinase known as wee-1.3(lz5) involved in regulating oocyte maturation, rescues this reduced hatch rate back to around 44% (Benton, et. al, 2021). In addition to interacting in the early embryo, previous research has shown that a pam-1 mutation rescues the loss of wee-1.3(RNAi) phenotype of precocious oocyte maturation (Benton, et. al, 2021). This project investigates if the suppressor mutation wee-1.3(lz5) can rescue the pam-1 meiotic defects. We compare time-lapse videos of pam-1 embryos, pam-1;wee-1.3(lz5) embryos, wildtype embryos, wildtype;wee-1.3(RNAi) embryos, and pam-1;wee-1.3(RNAi) embryos with GFP tagged histones in order to visual the chromosomes during meiosis. Our goal was to determine if wee-1.3 and pam-1 interact during meiosis to rescue the pam-1 meiotic defects, similarly to how they interact during oocyte maturation to rescue the wee-1.3 precocious oocyte maturation defects. In addition, we used cdk-1(RNAi) to inactive the cell cycle component CDK-1, which has been shown to result in a failure of meiotic divisions and a one-cell arrest phenotype in a wildtype worm (Van Der Voet, Lorson, Srinivasan, Bennett & Van Den Heuvel, 2009). CDK-1 is a component of the maturation promoting factor (MPF), which is regulated by WEE-1.3. Since we hypothesize that MPF activity may be lowered in pam-1 mutants, then we hypothesize that the pam-1 worms will be more sensitive to the inactivation of this component of MPF, however we saw no difference between pam-1 and wildtype strains when CDK-1 was inactivated. Thus far, the data suggests a possible partial rescuing of the pam-1 meiosis II defects with the wee-1.3(lz5) suppressor, but not a rescuing of the meiotic exit phenotypes. Our cdk-1 inactivation studies showed no interaction of pam-1 and cdk-1 during early embryo development. Previous studies have shown that active CDK-1 is required for entry into the M-phase, and degradation of CDK-1 is necessary to exit it. We propose the model that the wee-1.3(lz5) mutation causes overactive CDK-1, therefore improving the meiosis II timing of pam-1 mutants and not improving the pam-1 meiotic exit timing. This study provides better insights into the interactions of WEE-1.3, MPF, and PAM-1 during meiosis, which can lead us to better understanding the mechanism of PAM-1 in development.