Introduction of calcium buffers into the cytosol ofDictyostelium discoideumamoebae alters cell morphology and inhibits chemotaxis

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.

Section: Discussionmentioning

confidence: 99%

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

Furukawa

Maselli

Thomson

et al. 2003

“…It should, however, be noted that in the reported cAMP chemotaxis experiments, Dictyostelium speed was close to that observed during random motility, suggesting that stimulation was not at its maximal. In addition, introduction of EGTA calcium buffer stops net cell movement but does not prevent cell orientation in the direction of a cAMP gradient, revealed by protrusion elongation in this direction (Unterweger and Schlatterer, 1995;Van Duijn and Van Haastert, 1992). Intracellular loading with BAPTA, a calcium buffer which has a similar Ca 2+ dissociation constant to EGTA but an about 150-fold faster rate of Ca 2+ binding (Tsien, 1980), prevents both motility and oriented pseudopod emission (Schlatterer and Malchow, 1993;Unterweger and Schlatterer, 1995).…”

Section: Discussionmentioning

confidence: 99%

Calcium mobilization stimulatesDictyostelium discoideumshear-flow-induced cell motility

Fache

Dalous

Engelund

et al. 2005

Application of hydrodynamic mild shear stress to adherent Dictyostelium discoideum vegetative cells triggers active actin cytoskeleton remodeling resulting in net cell movement along the flow. The average cell speed is strongly stimulated by external calcium (Ca2+, K50%=22 μM), but the directionality of the movement is almost unaffected. This calcium concentration is ten times higher than the one promoting cell adhesion to glass surfaces (K50%=2 μM). Addition of the calcium chelator EGTA or the Ca2+-channel blocker gadolinium (Gd3+) transiently stops cell movement. Monitoring the evolution of cell-surface contact area with time reveals that calcium stimulates cell speed by increasing the amplitude of both protrusion and retraction events at the cell edge, but not the frequency. As a consequence, with saturating external calcium concentrations, cells are sensitive to very low shear forces (20 pN; σ=0.1 Pa). Moreover, a null-mutant lacking the unique Gβ subunit does not respond to external Ca2+ changes (K50%>1000 μM), although the directionality of the movement is comparable with that of wild-type cells. Furthermore, cells lacking the inositol 1,4,5-trisphosphate receptor (IP3-receptor) exhibit a markedly reduced Ca2+ sensitivity. Thus, calcium release from internal stores and calcium entry through the plasma membrane modulate cell speed in response to shear stress.

“…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.…”

Section: Role Of Ca 2+ In Flii-nmmiia Interactionsmentioning

confidence: 99%

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

Arora

Gregorio

et al. 2017

Fibroblasts remodel extracellular matrix collagen, in part, through phagocytosis. This process requires formation of cell extensions, which in turn involves interaction of the actin-binding protein flightless-1 (FliI) with non-muscle myosin IIA (NMMIIA; heavy chain encoded by MYH9) at cell-matrix adhesion sites. As Ca 2+ plays a central role in controlling actomyosin-dependent functions, we examined how Ca 2+ controls the generation of cell extensions and collagen remodeling. Ratio fluorimetry demonstrated localized Ca 2+ influx at the extensions of fibroblasts. Western blotting and quantitative (q)PCR showed high expression levels of the Ca 2+ -permeable transient receptor potential vanilloid-4 (TRPV4) channel, which co-immunoprecipitated with β1 integrin and localized to adhesions. Treatment with α2β1-integrin-blocking antibody or the TRPV4-specific antagonist AB159908, as well as reduction of TRPV4 expression through means of siRNA, blocked Ca 2+ influx. These treatments also inhibited the interaction of FliI with NMMIIA, reduced the number and length of cell extensions, and blocked collagen remodeling. Pulldown assays showed that Ca 2+ depletion inhibited the interaction of purified FliI with NMMIIA filaments. Fluorescence resonance energy transfer experiments showed that FliI-NMMIIA interactions require Ca 2+ influx. We conclude that Ca 2+ influx through the TRPV4 channel regulates FliI-NMMIIA interaction, which in turn enables generation of the cell extensions essential for collagen remodeling.