The Biochemical Basis for the Mechanics of Cytokinesis

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): Cytokinesis is a dramatic example of a cell shape change during which the mechanical constriction of the contractile ring leads to cell separation at the end of mitosis. Cytokinesis is essential for normal cell proliferation and is of medical interest for its role in hyperproliferative diseases such as cancer. In Dictyostelium discoideum, genetic interactions have been identified between two actin cross-linking proteins that have complementary cellular distributions. Cortexillin-I is localized to the contractile ring while dynacortin is cortically enriched but excluded from the contractile ring. The implication is that cells have evolved distinct actin cross-linking proteins with complementary cellular distributions that orchestrate cell shape changes, and these different actin cross-linking proteins may control regional cortical viscoelasticity. In this proposal, to ascertain how dynacortin controls viscoelasticity, its actin cross-linking mechanism will be studied using purified proteins and a variety of equilibrium and kinetic techniques. The cellular role of dynacortin will be studied using a variety of in vivo assays including dominant effects in wild type cells, suppression of cortexillin-I and rescue of a dynacortin loss-of-function mutant. Because cytokinesis is a mechanical process and the actin cytoskeleton is the principal contributor to the cell's viscoelasticity, we hypothesize that dynacortin and cortexillin-I control regional viscoelasticity. We are using laser-tracking microrheology to measure the viscoelastic moduli of interphase and dividing wild type and genetically engineered strains where dynacortin, cortexillin-I and other activities have been altered. Indeed, in preliminary experiments, dynacortin and cortexillin-I are significant modulators of cortical viscoelasticity. New genes involved in cortical shape control will be identified using genetic suppression of cortexillin-I and genetic enhancement of myosin-II. Myosin-II is the major mechanical force generator located at the contractile ring. One novel protein, DdERM, which was identified as a cortexillin-I suppressor, is hypothesized to tether the cortical actin to the plasma membrane and contribute to cortical viscoelasticity. DdERM is a fusion of two classes of mammalian actin-associated proteins, ezrin-radixin-moesin (ERM) and fimbrin. Thus, this molecule is of considerable interest because of its role in cortical function and cell shape control and its unusual domain structure.