Separation and identification of the major constituents of cytoplasmic gels from macrophages

Abstract

Proteins which bind to actin filaments in macrophages were investigated by developing a procedure for the isolation of cytoplasmic gels. The gels were found to consist of five major constituents: actin, filamin and the 105-kDa, 70-kDa and 55-kDa components. Prolonged exposure of this macromolecular complex to high-ionicstrength buffer solubilized almost all the proteins, leaving behind the 55-kDa component along with a large amount of actin. Gel filtration of the solubilized extract led to the isolation of five constituents comprising actin, filamin, the 105-kDa and 70-kDa polypeptides, plus a molecular species which eluted at the position of a 280-kDa globular protein. The biochemical and immunological properties of the 105-kDa component were analogous to those of a-actinin. Although several attempts were made to correlate the three other constituents (280-kDa, 70-kDa and 55-kDa) with known cytoskeletal proteins, their identity remains to be established. a-Actinin, and the 280-kDa and 70-kDa species all exhibited the ability to co-sediment with F-actin and to pack actin filaments into bundles. The bundling activity of the 70-kDa protein was significantly decreased in the presence of micromolar concentrations of calcium, while the activity of the 280-kDa protein was not. Such a Ca'+-sensitive protein could be very important in controlling the local cytoplasmic viscosity.It is now well established that most animal cells contain ordered arrays of three major fiber systems : microfilaments, microtubules and intermediate filaments. Because of their highly dynamic properties, microfilaments (actin filaments) are increasingly the subject of ultrastructural and biochemical investigations at the cellular level and in cytosolic extracts.Under appropriate conditions, cytosolic preparations from many cells can undergo a rapid transition from a soluble to a gel-like phase. This rise in viscosity is induced by a rapid polymerization of monomeric actin. Substantial variations in cytoplasmic consistency had also been observed during cell locomotion. It had been subsequently found that conditions which affect the viscoelastic properties of the cytoplasm, such as transient changes in free Ca", also influence actin-myosin interactions (for reviews see [l -31). These findings led Hellewell and Taylor [4] to propose that transitions from the sol to gel state may be directly involved in the generation of cytoplasmic movements.More recently, the use of techniques involving microinjection of fluorescently labeled proteins into living cells [S] has allowed a direct correlation to be established between the reorganization of actin filament bundles and motile activities in fibroblasts [6, 71. Therefore, the rapid assembly and disassembly of microfilaments is probably required for cell motility. The biochemical and structural bases for these dynamic cellular events are, however, very poorly understood. They are probably related to the properties of the actin molecule itself and also to its interactions with actin-associated proteins. Ma...

Lysosomal movements during heterophagy and autophagy: With special reference to nematolysosome and wrapping lysosome

“…It is generally considered that the gel-sol transformation of actin is mediated by actinassociated proteins. Several kinds of actin-associated proteins such as gelsolin Yin et al, 19811, a-actinin (Bennett et al, 19841, actinbinding protein (Hartwig and Stossel, 19751, and actinbundling protein (Pacaud, 1986;Pacaud and Harricane, 1987) were isolated from the cytoplasm of macrophages and characterized. Some of these actinassociated proteins were considered t o be involved in the gel-sol transformation of actin in the cytoplasm of macrophages (Hartwig and Stossel, 1975;Pacaud, Fig.…”

“…The interaction of actin with actin-associated proteins was reported to be sensitive to intracellular calcium ions, which are a second messenger on many cell functions (Bennett et al, 1984;Pacaud, 1986;Pacaud and Harricane, 1987;Stossel et al, 1985;. Since the intracellular concentration of calcium ion was connected with the ligand-receptor interaction and endocytosis (Young et al, 19841, the change of calcium ion concentration at micromolar level might play a role as a signal for regulation of the lysosomal movement following endocytosis during heterophagy.…”

In situ and in vitro morphological evidence for the interaction of actin filaments with lysosomes in rat alveolar macrophages.

“…Furthermore, there is some evidence indicating that lysosomal membranes in polymorphonuclear leukocytes contain intramembranous particles (14) and glycoproteins associated with their membranes (16). The interaction of actin with actin-associated proteins was reported to be sensitive to intracellular calcium ion which is a second messenger in cell functions (4,17,18,22,31,32). It was reported that the ligand-receptor interaction increased the concentration of intracellular calcium ion, and increasing calcium ion in cytoplasm might play a role of the signals for endocytic process (33).…”

“…Several kinds of actinassociated proteins were isolated from nonmuscle cells (22). Actin-associated proteins such as gelsolin (31), a-actinin (4), actin-binding protein (10) and actin-bundling protein (17,18) were isolated from the cytoplasm of macrophages. These actinassociated proteins are thought to be involved in the gel-sol transformation of actin in the cytoplasm of macrophages (10, 17, 18, 22-24, 31, 32).…”

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

“…The macrophage protein was shown to bind to polymerized actin in a calcium-dependent manner (13,14). Also, Matsushima and co-workers (12) have purified L-plastin from human macrophages (identified as L-plastin by sequence [8]) and have demonstrated that it is phosphorylated at serine residues.…”