Affiliation:
1. Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA
Abstract
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 ΔEF2) 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 ΔEF2 protein. The actin-binding activity of the 34 kDaΔEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa ΔEF2 proteins were expressed in 34-kDa-null and 34 kDa/α-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa ΔEF2 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 ΔEF2. 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.
Publisher
The Company of Biologists
Reference63 articles.
1. Andre, B., Noegel, A. A. and Schleicher, M.(1996). Dictyostelium discoideum contains a family of calmodulin-related EF-hand proteins that are developmentally regulated. FEBS Lett.382,198-202.
2. Brier, J., Fechheimer, M., Swanson, J. and Taylor, D. L.(1983). Abundance, relative gelation activity, and distribution of the 95,000 dalton actin-binding protein from Dictyostelium discoideum.J. Cell Biol.97,178-185.
3. Clarke, M., Bazari, W. L. and Kayman, S. L.(1980). Isolation and properties of calmodulin from Dictyostelium discoideum.J. Bacteriol.141,397-400.
4. Condeelis, J. S. and Taylor, D. L. (1977). The contractile basis of amoeboid movement. V. The control of gelation, solation,and contraction in extracts from Dictyostelium discoideum.J. Cell Biol.74,901-927.
5. Cubitt, A. B., Firtel, R. A., Fischer, G., Jaffe, L. F. and Miller, A. L. (1995). Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaquorin. Development121,2291-2301.
Cited by
59 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献