title: Calcium control of macrophage cytoplasmic gelation: evidence for the involvement of the 70,000 Mr 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.
authors.affiliation: Centre de Recherches de Biochimie Macromoléculaire CNRS, LP 8402, INSERM, U 249, Université de Montpellier 1, France.
authors.forename: M
authors.initials: M
authors.lastname: Pacaud
authors.forename: M C
authors.initials: MC
authors.lastname: Harricane
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects*; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D003599:Cytoskeleton / Q000648:ultrastructure; D005782:Gels; D018260:Gelsolin; D008264:Macrophages / Q000187:drug effects* / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D008854:Microscopy, Electron; D011817:Rabbits; D012995:Solubility
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D005782:Gels; D018260:Gelsolin; D008840:Microfilament Proteins; C052523:actin filament bundling proteins; D002118:Calcium