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Regulation of Cytokinesis by Two Essential Proteins Cofilin and Pkd2

Malla, Mamata
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1667151729535541

Abstract Details

2022, Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology).
Cytokinesis by most eukaryotic cells depends on the actomyosin contractile ring which drives the ingression of the cleavage furrow and the daughter cell separation with the necessary mechanical force. The regulation of actin cytoskeletal structures thus play an essential role in cytokinesis. This is aided by many other essential molecules including the putative ion channel Pkd2 that our lab recently discovered. In this study, I dissected the contributions by an essential actin filament-severing protein cofilin and Pkd2 to cytokinesis through quantitative microscopy and yeast genetics in the unicellular model organism Schizosacchacromyces pombe. In the first part of my dissertation, I used GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live cells. Lifeact is a small peptide which does not interfere with the function of actin at low concentration (Courtemanche et al., 2016). Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min during ring assembly and maturation to a peak number of ~198,000, which was followed by a loss of actin at 5,400 molecules/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that has highly reduced actin filament severing activity, contractile rings accumulated polymerized actin at twice the normal rate resulting in ~80% more actin at the peak and proportional increases in type II myosins Myo2, Myp2 and formin Cdc12. About 30% of adf1-M3 mutant cells failed to constrictiv their rings fully, whereas the rest lost actin from the rings at almost the wild-type rates. Surprising, cells with mutations of type II myosins, myo2-E1 and myp2∆, had contractile rings with only half the normal actin filaments and slow rates of actin loss. My results demonstrate the importance of directly quantifying actin molecules in contractile rings and the contributions of cofilin and type II myosins to the contractile ring constriction. In the second part of my dissertation, I focused my work on understanding another essential cytokinetic protein identified by our lab, the fission yeast polycystin, Pkd2. Polycystins are a family of conserved calcium ion channels, mutations of which lead to human genetic disorder Autosomal Dominant Polycystic Kidney Disease. Schizosacchromyces pombe possesses a single essential polycystin Pkd2 that localizes to the plasma membrane and is required for cell proliferation (Sinha et al., 2022). I carried out a functional analysis of Pkd2 based on its Alphafold predicted structure which consisted of an N-terminal lipid-binding (LBD), a central transmembrane (TMD) and a C-terminal cytoplasmic (CCD) domains. LBD assumes a unique immunoglobulin-fold, while TMD contains nine transmembrane helices. Both were essential. Although the mostly disordered CCD was not, its removal led to clustering of Pkd2 in eisosomes, a microdomain of the plasma membrane. Inhibiting eisosome assembly prevented the clustering, while disrupting ER-PM contacts further increased it. Pkd2 shared similar structure with two other putative channels Trp663 and Trp1322, but their intracellular localization and function diverged from each other. The Pkd2 ortholog from S. japonicus fully replaced Pkd2. My work suggests a potential mechanism of regulating polycystin function through its clustering in the furrowing microdomains of the plasma membrane. In summary, I studied cofilin and Pkd2, two essential proteins which regulate cytokinesis in fission yeast. Cofilin stabilizes the contractile ring and makes the ringv constriction process efficient. The different domains of Pkd2 have unique contributions to its intracellular localization and function. Finally, my finding that Pkd2 clusters on the cell surface is exciting suggesting a novel way of regulating polycystin function.
Qian Chen (Committee Chair)
Biology; Cellular Biology; Molecular Biology

Recommended Citations

Citations

  • Malla, M. (2022). Regulation of Cytokinesis by Two Essential Proteins Cofilin and Pkd2 [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1667151729535541

    APA Style (7th edition)

  • Malla, Mamata. Regulation of Cytokinesis by Two Essential Proteins Cofilin and Pkd2. 2022. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1667151729535541.

    MLA Style (8th edition)

  • Malla, Mamata. "Regulation of Cytokinesis by Two Essential Proteins Cofilin and Pkd2." Doctoral dissertation, University of Toledo, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1667151729535541

    Chicago Manual of Style (17th edition)

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© 2022, some rights reserved.Creative Commons License Regulation of Cytokinesis by Two Essential Proteins Cofilin and Pkd2 by Mamata Malla is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Toledo and OhioLINK.
Release 3.2.12