Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium
2003
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
[ 45 Ca]CaCl 2 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 kDaΔ
EF2 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.
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