Actin gelation and structure of cortical cytoplasm.
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Biomedical subjects
J H Hartwig
Publications and source records attributed to J H Hartwig.
Ca2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization.
Gelsolin complexes with calcium (gelsolin-Ca2+) binds to the ends of actin filaments to which monomers add preferentially during elongation. It forms a stable complex with actin in a low ionic strength solution which does not normally favor the polymerization of actin. Gelsolin-Ca2+ increases the rate of nucleation of actin which precedes polymerization, but decreases the rate of elongation of the filaments. The final average length of filaments formed in the presence of gelsolin-Ca2+ is shorter and the equilibrium monomer concentration increases relative to actin polymerized in the absence of gelsolin-Ca2+. Gelsolin-Ca2+ also increases the number of actin filaments because the magnitude of the increase in monomer concentration is disproportionately small compared with the reduction in polymer length. In these respects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of ects, the population of actin filaments formed during polymerization in the presence of gelsolin-Ca2+ is similar to that resulting from the action of gelsolin on previously assembled actin filaments (Yin, H. L., Zaner, K. S., and Stossel, T. P. (1980) J. Biol. Chem. 255, 9494-9500). The calcium-dependent shortening of actin filaments is the primary mechanism for the dissolution of an actin gel by gelsolin. Therefore, the ability of gelsolin to produce short filaments irrespective of the initial state of assembly of the actin offers flexibility for controlling the network structure of the cytoplasm in which either the monomeric or polymeric form of actin molecules might predominate at different times.
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Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments.
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Distribution of actin-binding protein and myosin in polymorphonuclear leukocytes during locomotion and phagocytosis.
Polymorphonuclear (PMN) leukocytes, highly motile ameboid cells vital for mammalian defense against infection, acquire a distinct polarized morphology during locomotion and phagocytosis. An organelle-excluding pseudopod extends in the direction of movement and surrounds objects during phagocytosis. The anterior pseudopod contains a three-dimensional network of actin filaments. Actin-binding protein (ABP) and myosin cause the crosslinking and contraction, respectively, of actin filaments in vitro. We used indirect immunofluorescence to study the redistribution of myosin and ABP molecules in rabbit PMN leukocytes during locomotion and phagocytosis. In unpolarized PMN leukocytes, ABP and myosin had a diffuse distribution with some predilection for the cortex. In polarized PMN leukocytes crawling toward yeast particles, myosin and ABP staining concentrated in the anterior pseudopod. In PMN leukocytes fixed during phagocytosis of the yeast particles, antimyosin and anti-ABP staining concentrated strikingly in the distal portions of the pseudopod embracing the yeasts. Staining for catalase, a cytoplasmic protein in PMN leukocytes, for lactoferrin, a protein of specific granules, and for myeloperoxidase, a protein of azurophilic granules, was not concentrated in pseudopods. Taken together with available morphologic and biochemical information, these findings are consistent with a mechanism wherein interactions of actin, ABP and myosin redistribute cortical cytoplasm into pseudopods involved in locomotion and phagocytosis.
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Cytochalasin B and the structure of actin gels. II. Further evidence for the splitting of F-actin by cytochalasin B.
Cytochalasin B decreased the flow birefringence and s20,w and increased the extinction angle of actin filaments in salt solutions favoring polymerization of the protein. These changes occurred without a detectable increase in the equilibrium actin monomer concentration determined by a radioassay. These results complement earlier observations indicating that cytochalasin B shortens actin filaments without net depolymerization. Analyzed in terms of Flory's classical network theory, this shortening accounts for the marked effect of cytochalasin B in dissolving the gel structure of F-actin crosslinked by actin-binding protein concentrations near the critical concentration for incipient gelation. Cytochalasin B decreased the annealing rate of low concentrations of actin filament fragments prepared by sonic disruption. The result is consistent with the idea that cytochalasin B binds to the ends of actin filaments, and may explain how cytochalasin B causes filament shortening.
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Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis.
Actin-binding protein (ABP) and myosin are proteins that influence the rigidity and movement, respectively, of actin filaments in vitro. We examined the distribution of ABP and myosin molecules in acetone-fixed rabbit lung macrophages by means of immunofluorescence. The staining for both of these proteins in unspread cells was quite uniform, but was reduced in the nucleus and concentrated slightly in the periphery. The peripheral accumulation of staining attenuated in uniformly spread cells, although filopodia and hyaline veils definitely stained. In cells fixed during ingestion of yeast particles, the brightest staining correlated with the disposition of organelle-excluding pseudopodia initially surrounding the yeast. After phagocytosis was complete and the yeasts resided in intracellular vacuoles, no concentration of staining around the ingested yeasts was detectable. We conclude that ABP and myosin molecules are components of the structural unit of the cell responsible for spreading and phagocytosis, the hyaline cortex, a region known to be rich in actin filaments. The findings are consistent with the theory that these molecules control the rigidity and movement of filaments in the periphery of the living macrophage.
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Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.
Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.
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The motor of amoeboid leucocytes.
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Cytochalasin B and the structure of actin gels.
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The gelation of actin by actin-binding protein.
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Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation.
Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of beta-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.
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Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.
Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.
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Interactions of actin, myosin, and an actin-binding protein of rabbit pulmonary macrophages. III. Effects of cytochalasin B.
Low concentrations (greater than or equal to 10(-7) M) of cytochalasin B reversibly inhibit the temperature-dependent gelation of actin by an actin-binding protein. The cytochalasin B concentrations which maximally inhibit actin gel formation are 10-fold lower than the concentrations which maximally impair phagocytosis by intact macrophages. Cytochalasin B also prevents the polymerization of monomeric actin in sucrose extracts of macrophages in the absence but not the presence of 0.1 M CKl. 10(-6) M cytochalasin B dissolves macrophage extract gels and gels comprised of purified actin and actin-binding protein by dissociating actin-binding protein from actin filaments. This concentration of cytochalasin B, however, does not depolymerize the actin filatments.
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Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages.
Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.
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Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.
The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity. In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.
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