Studies of the Actin Binding Activity of Dictyostelium discoideum Myosin II Heavy Chain Kinase A.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Mary Elizabeth Keener (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Paul Steimle

Abstract: Dictyostelium discoideum is a primitive, eukaryotic organism that relies on myosin II and actin contraction for cytokinesis, migration, and other important cellular processes. In order for contraction to occur, myosin II monomers must first assemble into bipolar filaments. In Dictyostelium, bipolar filament assembly is negatively regulated by myosin heavy chain (MHC) phosphorylation. Three kinases have been shown to catalyze MHC phosphorylation (MHK-A, -B, -C), driving myosin II disassembly. Of the three, MHK-A is the most extensively studied. Structurally, MHK-A is comprised of three functional domains: an N-terminal coiled coil region (CC), a catalytic domain, and a WD repeat domain. The CC domain has been shown, in vitro, to bind and bundle actin filaments and inhibit myosin II binding to F-actin. The studies described in this thesis focus on two aims: 1) to understand the role of the CC domain in reorganizing myosin II and actin within the cell and 2) to determine if intramolecular interactions between the CC and WD repeat domains within the MHK-A molecule are involved in regulating MHKA’s activity. To address the first aim, the triton cytoskeleton ghost isolation assay was conducted on Ax2, Ax2 + GFP-CC, Ax2 + GFP-C800A, MHK-A null, and MHK-A null + GFP-CC cell lines containing various truncations to determine the analyze the effects of over-expressing the CC domain on myosin II and F-actin organization within the cell. Complementary localization studies were also conducted involving Ax2 wild-type cells and GFP-tagged cells over-expressing the CC domain (GFP-CC) to visually determine myosin II and actin localization within the cell via confocal imaging. Results from the triton cytoskeleton studies indicate that over-expression of the CC domain in the Ax2 cells leads to an increase in cytoskeletal myosin II. MHK-A null and MHK-A null cells over-expressing the CC domain showed a significant increase in cytoskeletal myosin II in comparison to the Ax2 cells. However, the CC domain had no apparent effect in the absence of endogenous MHK-A as indicated by the relatively equal amounts of myosin II in both MHK-A null and MHK-null + GFP-CC pellet fractions. Ax2 + GFP-C800A cells exhibit essentially the same level of cytoskeletal myosin II compared to that of the Ax2 cells. Localization studies revealed increased amounts of cytoplasmically localized myosin II aggregates in the Ax2 + GFP-CC cells in comparison to the Ax2 cells. Also, cells over-expressing the CC domain appear to have more cytoplasmic F-actin than the Ax2 cells. Furthermore, the actin appears to be more aggregated in the over-expressed CC cells. To address the second aim, purified CC and GSTtagged WD repeat domains were subjected to a GST “pull-down” assay and Western blot to determine if an interaction between the domains exists. Results from these experiments revealed no interaction between the WD repeat and CC domains, evident by the absence of CC in the GST-WD bead-associated fraction. This study shows that the CC domain plays an integral role in reorganizing the actin-myosin cytoskeleton and that the mechanisms regulating the full-length MHK-A do not appear to be determined by WD repeat and CC domain interactions.

Additional Information

Language: English
Date: 2008
Myosin, Kinase, Dictyostelium, Actin, Cytokinesis, MHC
Dictyostelium discoideum $x Physiology.
Cytokinesis $x Physiology.
Cytoskeletal proteins.
Protein kinases.

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