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doi: 10.1242/10.1242/jcs.00220

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Journal of Cell Science 116, 187-196 (2003)
doi: 10.1242/jcs.00220

Research Article

Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium

Ruth Furukawa*, Andrew Maselli*, Susanne A. M. Thomson, Rita W. L. Lim, John V. Stokes and Marcus Fechheimer{ddagger}

Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA

{ddagger} Author for correspondence (e-mail: fechheim{at}cb.uga.edu)

Accepted 14 October 2002

The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa {Delta}EF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [45Ca]CaCl2 revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 µM. This calcium binding is absent in the 34 kDa {Delta}EF2 protein. The actin-binding activity of the 34 kDa {Delta}EF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa {Delta}EF2 proteins were expressed in 34-kDa-null and 34 kDa/{alpha}-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa {Delta}EF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa {Delta}EF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

Key words: Cytoskeleton, Motility, EF hands, Calcium regulation, Actin-binding protein, Dictyostelium




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