Date of Award
Spring 2025
Document Type
Thesis (Ph.D.)
Department or Program
Biochemistry and Cell Biology
First Advisor
James Moseley
Second Advisor
Erik Griffin
Third Advisor
Arminja Kettenbach
Abstract
Every dividing eukaryotic cell must coordinate cytokinesis with mitosis in both space and time to ensure that each daughter cell receives equal complements of the duplicated genetic material. This process is crucial for maintaining proper growth and development in many organisms. The importance of cytokinesis is further supported by the finding that its failure causes polyploidy, which, in turn, can result in defective mitoses and chromosomal instability. These defects have varying degrees of cellular consequences and have been linked to the development of various diseases like cancer. Nearly one billion years ago, the molecular mechanism underlying cytokinesis through a contractile actomyosin ring originated in a common ancestor of amoebas, fungi, and animals. These organisms share much of the same cytokinetic machinery, meaning that knowledge gained from studying the well characterized model organism Schizosaccharomyces pombe will provide important mechanistic insights behind human cellular controls. While many of the individual components of the CAR have been identified, we do not fully understand how they are regulated and time and space and how this contributes to the fidelity of cytokinesis. My thesis work focused on understanding how protein phosphorylation allows cytokinetic proteins to acquire temporal and spatial specificity. My work identified the highly conserved protein phosphatase PP2A-B56 promotes efficient cytokinesis through the anillin-like protein Mid1. We found that cells lacking Par1 had reduced levels of cellular Mid1, resulting in cytokinetic defects. We were able to rescue these cytokinetic defects by restoring the levels of Mid1 in par1 cells, thereby solidifying the connection between PP2A-B56, Mid1 abundance, and cytokinesis efficiency. Previous groups have shown that increased Mid1 levels also lead to cytokinetic defects. This requirement for regulating Mid1 expression could represent a conserved principle because altered levels of the Mid1-related protein anillin in higher eukaryotes results in similar cytokinetic failure. Next, I became interested in the conserved protein kinase Pdk1 and its potential role in regulating cytokinesis. Pdk1 is considered a ‘master’ kinase because of its ability to phosphorylate and activate more than 23 downstream protein kinases that are involved in signaling pathways ranging from actin organization, inflammation, cell adhesion and motility. Unsurprisingly, the dysregulation of Pdk1 has been associated with numerous cancers and is believed to lead to poor clinical outcomes. Previous studies performed in fission yeast suggest that cells lacking Pdk1 have numerous cytokinetic defects, including delayed contractile actomyosin ring assembly and diffuse Mid1 localization. My thesis work aimed to further characterize these defects and to understand if Pdk1 has an additional role in regulating cytokinesis. In the absence of Pdk1, we found that cells exhibit cytokinetic defects that likely arise from structural issues with the contractile actomyosin rings. We also have identified a potential role of Pdk1 in regulating the assembly of eisosomes through phosphorylation of Pil1. Collectively, this thesis considers how spatial and temporal control is generated within a defined biological system. These results have the potential to uncover principles that operate in higher eukaryotes across varying scales of time and space.
Recommended Citation
Chrupcala, Madeline L., "Phospho-regulation of cytokinetic proteins in fission yeast" (2025). Dartmouth College Ph.D Dissertations. 406.
https://digitalcommons.dartmouth.edu/dissertations/406
