Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton inDictyostelium

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.

Requirements for Hirano Body Formation

“…Therefore, it has been proposed that CTinduced model Hirano body formation may be the result of increased F-actin binding and loss of calcium regulation (16). Further studies using additional mutated forms of the 34-kDa protein are consistent with the conclusion that activation of actin binding and loss of calcium regulation are biochemical properties invariably associated with induction of model Hirano bodies (17,25). On the cellular level, this model system was used to demonstrate that Hirano body formation proceeds by formation of small aggregates, followed by consolidation into a single large Hirano body (26).…”

Cancer cells become less deformable and more invasive with activation of β-adrenergic signaling

“…It is possible that the effects of Ca 2+ on cell mechanotype might be mediated through cytoskeletal actin, whose organization and dynamics are regulated by this divalent cation (Dushek et al, 2008;Young et al, 1994). The function and activity of actin-associated proteins that regulate crosslinking and severing also depend on Ca 2+ (Furukawa et al, 2003;Witke et al, 1993;Yamamoto et al, 1982). For example, the Ca 2+ -calmodulin complex binds to filamin, which is an actin crosslinker that regulates the cell mechanical phenotype (Kasza et al, 2009;Nakamura et al, 2005;Stossel et al, 2001).…”

TRPV4 mediates the Ca2+ influx required for the interaction between flightless-1 and non-muscle myosin, and collagen remodeling

“…These processes in turn influence cell shape, endocytosis, morphogenesis, locomotion, growth and division (Furukawa et al, 2003). For example, in Dictyostelium, Ca 2+ fluxes derived from extracellular Ca 2+ or intracellular Ca 2+ stores are required for cell spreading in locomotion and chemotaxis (Furukawa et al, 2003;Unterweger and Schlatterer, 1995).…”

“…These processes in turn influence cell shape, endocytosis, morphogenesis, locomotion, growth and division (Furukawa et al, 2003). For example, in Dictyostelium, Ca 2+ fluxes derived from extracellular Ca 2+ or intracellular Ca 2+ stores are required for cell spreading in locomotion and chemotaxis (Furukawa et al, 2003;Unterweger and Schlatterer, 1995). Consistent with these data and the notion that FliI is a Ca 2+ -dependent actin-capping protein, we found that when FliI WT cells were loaded with BAPTA-AM to clamp [Ca 2+ ] i , the association of FliI with NMMIIA, the formation of cell extensions and collagen compaction were all inhibited.…”