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Reorganization of the actomyosin cytoskeleton of the one-cell C. elegans embryo is necessary to set up the anterior-posterior axis. Polarity is established in part through cortical flow that requires numerous cytoskeletal proteins to control cortical dynamics. PAM-1 encodes a puromycin-sensitive aminopeptidase required for proper regulation of the cortex. Our objective is to determine the mechanism by which PAM-1 regulates the cytoskeleton. pam-1 mutants display phenotypic defects consistent with a misregulation of the actin cytoskeleton. First, protein localization of moesin is slower to clear from the posterior than in wildtype embryos and the foci are smaller and sparser. Second, non-muscle myosin, NMY-2 appears to have a less robust network of puncta around the cortex and is slower to clear from the posterior than in wildtype embryos. In wildtype embryos, both moesin and NMY-2 puncta are spread throughout the cortex and after polarization is initiated, they localize to the anterior side of the embryo and concentrate at the cleavage furrows of dividing cells. Lastly, pam-1 mutants show defects in the regulatory processes leading up to cytokinesis, such as an over-active cortex, weak or no pseudocleavage, and blebbing at the cleavage furrow. These defects suggest the cortical cytoskeleton is not properly regulated and could be due to other cytoskeletal proteins interacting with PAM-1 to regulate cortical dynamics. Anillin, encoded by the ani-1 gene, functions by remodeling the actin cytoskeleton. It binds non-muscle myosin and is suggested to be a key regulator alongside PAM-1 in regulating cortical activity. By using RNA interference on ani-1 in both wildtype and pam-1 mutants, we propose a model for the interaction between the two proteins suggesting PAM-1 regulates cortical activity downstream of ANI-1. However, while PAM-1 has been shown to be required for large NMY-2 foci size, it does not affect puncta count; whereas ANI-1 is required for the number of NMY-2 foci organized suggesting PAM-1 and ANI-1 work separately to organize NMY-2 prior to cytokinesis. We also identified phenotypic defects in pam-1 mutants resulting in improper organization of moesin and NMY-2 around the cortex, using fluorescence tagging. Our studies reveal possible mechanisms by which PAM-1 regulates anterior-posterior axis polarization and suggest interactions between known cytoskeletal proteins and PAM-1 in organizing the actomyosin cytoskeleton.
Tesone, Zoe, "The PAM-1 Aminopeptidase Interacts With Cytoskeletal Regulators in Controlling Cortical Dynamics in the One-Cell C. elegans Embryo" (2021). Biology Honors Papers. 41.