Calcium control of macrophage cytoplasmic gelation: Evidence for the involvement of the 70000Mr actin-bundling protein

Abstract

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin

“…Actin-severing proteins, such as gelsolin and villin, are known to reduce the viscosity and length of actin filaments in vitro in the presence of micromolar Ca2+ (Kwiatkowsky et al., 1989;Mooseker et al, 1980;Bretscher & Weber, 1980a). This observation thus confirms our previous suggestion that p70 is not capable of fragmenting actin filaments (Pacaud & Harricane, 1987).…”

“…The selective and quantitative release of L-plastin, from macrophage-insoluble cytoplasmic gels, by submicromolar concentrations of free Ca2+ was previously reported (Pacaud & Harricane, 1987). The data presented here indicated that Ca2+ binding to the purified protein, in the physiologically important concentrations, leads to a loss in its ability to associate with actin filaments.…”

“…Half-maximal inhibition occurred at 1.6 pM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 mM.…”

“…Functional Significance of L-Plastin. According to our estimates, there is approximately one molecule of L-plastin per seven or eight actin monomers in macrophage cytosolic fractions, whereas a maximum of three molecules of plastin can bind to three actin monomers in a filamentous form (Pacaud & Harricane, 1987). Thus, a large fraction of this protein might well be associated with F-actin in resting macrophages.…”

“…We were subsequently able to demonstrate that a rise in the free calcium concentration of cytosolic extracts, within the 0006-2960/93/0432-3448504.00/0 physiological range, could trigger sequential changes in the protein composition of actin gels (Pacaud & Molla, 1987). Further studies on isolated cytoplasmic gels and purified 70-kDA protein led us to suggest that Ca2+ control of the interactions between this protein and F-actin may exert a dominant influence in regulating the state of microfilament organization (Pacaud & Harricane, 1987). Despite the established view that non-muscle a-actinin is a calciummodulated protein (for a review see Pollard and Cooper (1986), calcium had no effect on the association of a-actinin with F-actin in cytoplasmic gels (Pacaud & Molla, 1987).…”