Abstract
Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.
Highlights
Cytokinesis is an elegant cellular process because both contractile force generation and new actin filament polymerization act in concert to produce the desired dynamic shape changes
We describe the purification of the native dynacortin complex from D. discoideum and demonstrate that it is homomeric
Purification of Native D. discoideum and Recombinant Dynacortin—In our previous study, we determined that native D. discoideum dynacortin separated on a size exclusion column with a large apparent Stokes radius of about 5.7 nm [3]
Summary
Cytokinesis is an elegant cellular process because both contractile force generation and new actin filament polymerization act in concert to produce the desired dynamic shape changes. The second module is a global pathway that is controlled by the RacE small GTPase and includes dynacortin and the actin bundling protein coronin [3, 7, 8]. Three of these proteins, RacE, myosin-II, and cortexillin I, have been shown to contribute to the bending modulus and/or surface tension ( in-plane elasticity) of the cell (9 –11). The recombinant protein has an identical Stokes radius to the native D. discoideum complex and directly binds and bundles actin filaments into complex. All of our data indicate that cells have evolved different actin cross-linkers that localize in distinct regions of the cell but that collaborate to promote complex cell shape changes
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