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J H Hartwig

Publications and source records attributed to J H Hartwig.

At least 91 records · Page 5Linked to original sources
PubMed1988

The organization and regulation of the macrophage actin skeleton.

To move, leukocytes extend portions of their cortical cytoplasm as pseudopods. These pseudopods are filled with a three-dimensional actin filament skeleton, the reversible assembly of which in response to receptor stimulation is thought to play a major role in providing the mechanical force for these protrusive movements. The organization of this actin skeleton occurs at different levels within the cell, and a number of macrophage proteins have been isolated and shown to affect the architecture, assembly, stability, and length of actin filaments in vitro. The architecture of cytoplasmic actin is regulated by proteins that cross-link filaments in higher-order structures. Actin-binding protein plays a major role in defining network structure by cross-linking actin filaments into orthogonal networks. Gelsolin may have a central role in regulating network structure. It binds to the sides of actin filaments and severs them, and binds the "barbed" filament end, thereby blocking monomer addition at this end. Gelsolin is activated to bind actin filaments by microM calcium. Dissociation of gelsolin bound on filament ends occurs in the presence of the polyphosphoinositides, PIP and PIP2. Calcium and PIP2 have been shown to be intracellular messengers of cell stimulation.

Actins↗
PubMed1988

The structure of the macrophage actin skeleton.

The actin skeleton of the macrophage consists of a three-dimensional network of actin filaments and associated proteins. The organization of this multiprotein structure is regulated at several levels in cells. Receptor stimulation induces a massive actin polymerization at the cell cortex, changes in cell shape and active cellular movements. Gelsolin may have a pivotal role in restructuring the actin skeleton in response to agonist stimulation, as the activity of this potent actin-modulating protein is regulated by both Ca2+ and polyphosphoinositides. Micromolar concentrations of Ca2+ activate gelsolin to bind to the sides of actin filaments, sever, and cap the filament end. Polyphosphoinositides, in particular PIP and PIP2, release gelsolin from the filament ends. A structure-function analysis of gelsolin indicates that its N-terminal half is primarily responsible for severing actin filaments, and elucidates mechanisms by which Ca2+ and phospholipid may regulate gelsolin functions. The ultrastructure of actin filaments in the macrophage cortical cytoplasm is regulated, to a large extent, by the actin cross-linking protein, actin-binding protein (ABP) which defines filament orthogonality.

Actins↗
PubMed1987

Vasopressin-induced changes in the three-dimensional structure of toad bladder apical surface.

The apical plasma membrane of toad bladder granular cells undergoes a rapid and dramatic increase in water permeability in response to vasopressin stimulation. Previous studies have shown that this permeability increase is accompanied by characteristic changes in the morphology of this membrane and that these changes may be involved in the hormonal response. In this report, we have used the technique of rapid freezing and freeze drying to obtain high resolution stereo images of the surface of the granular cell apical plasma membrane before and during vasopressin stimulation. Using this approach, we confirmed that vasopressin induces a ridge-to-villus transformation of the cell surface even in the absence of osmotic water flow, but now show that this transformation occurs at least in part via a retraction of segments of preexisting ridges, rather than by the growth of new microvilli from the apical cell surface. This is also demonstrated by the finding that vasopressin induces the ridge-to-villus transformation of the cell surface even in the presence of cytochalasin D. In addition, the rapid-freeze, freeze-dry technique reveals that the surface glycocalyx of the epithelial cells consists of a complex, three-dimensional network of filaments that is heterogeneous among different cells. Finally, vasopressin-induced tubular invaginations of the apical plasma membrane were visualized in stereomicrographs, and the number and size of such invaginations were altered in the presence of cytochalasin D. These may represent surface images of vasopressin-induced exo- and endocytotic events that are related to membrane permeability changes.

Animals↗
PubMed1986

Actin filament architecture and movements in macrophage cytoplasm.

Actin filaments are the predominant structural elements in macrophage cortical cytoplasm. These fibres form a unique orthogonal network that fills all lamellae extended from the cell and which, in the cell body, bifurcates to form layers 0.2-0.5 micron thick on the cell top and bottom. Single short filaments, 0.1 micron in length, intersect in space in either T-shaped or X-shaped overlaps to form this ultrastructure. Network assembly and pseudopod extension occur when actin filaments within the network elongate. This filament growth is driven by a large storage pool of actin bound to the sequestering protein, profilin. Elongation is regulated by acumentin, gelsolin and possibly severin, proteins that bind to the end of the filaments, preventing the addition of actin monomers to the filaments. The cytosolic concentration of calcium controls whether filaments assemble or disassemble. Filaments can assemble when the filament ends are not blocked by gelsolin, a condition predicted to occur when the calcium concentration is less than 0.1 micron. Orthogonality results when actin filaments are cross-linked by molecules of actin-binding protein.

Actins↗
PubMed1986

The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages.

A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin-binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)

Actin Cytoskeleton↗
PubMed1984

Identification of actin-binding protein and villin in toad bladder epithelia.

Two actin-modulating proteins, actin-binding protein and villin, have been identified and functionally characterized in toad bladder epithelial cells. A 270-kdalton protein from bladder epithelial extracts 1) cross-reacts with antibodies to purified toad oocyte or rabbit macrophage actin-binding protein and comigrates with these proteins on gel electrophoresis, 2) contains all of the actin filament cross-linking activity found in epithelial cell extracts, and 3) its activity is calcium insensitive. A 95-kdalton protein from the same extracts 1) cross-reacts with antibodies to purified toad oocyte villin and comigrates with purified villin from the oocyte and chicken intestine on polyacrylamide gels, and 2) is capable of decreasing the viscosity of actin solutions only in the presence of calcium. These proteins offer potential sites for calcium-dependent and -independent regulation of toad bladder epithelial cell structure.

Animals↗
PubMed1984

The motor of leukocytes.

The movements of leukocytes involve extension, flow, and contraction of a margin of organelle-excluding cytoplasm. Actin is the principal structural component of this region. This paper reviews evidence that the expansion of cortical cytoplasm can result from the growth of actin polymers into an orthogonal network in which actin fibers branch perpendicularly under the influence of actin-binding protein. Flow occurs when actin filaments are disassembled and severed. The assembly and fragmentation of actin are regulated by actin-modulating proteins such as profilin, which sequesters actin monomers, acumentin, which binds to the slow-growing end of actin fibers, and gelsolin, a calcium-regulated protein that binds to the fast-growing end of actin polymers and severs actin filaments. Contraction of the actin network is caused by myosin, the assembly and activity of which are regulated by its state of phosphorylation, which is in turn controlled by phosphorylating and dephosphorylating enzymes and by calmodulin and calcium. Present information leads to the prediction that intracellular calcium gradients guide cytoplasmic movement and that the direction of actin assembly and therefore of cytoplasmic extension is toward regions of low cytoplasmic free calcium.

Actins↗
PubMed1983

Isolation and some structural and functional properties of macrophage tropomyosin.

Tropomyosin purified from rabbit lung macrophages is very similar in structure to other nonmuscle cell tropomyosins. Reduced and denatured, the protein has two polypeptides which migrate during electrophoresis in sodium dodecyl sulfate on polyacrylamide gels with slightly different mobilities corresponding to apparent Mr's of about 30 000. Following cross-linking by air oxidation in the presence of CuCl2, electrophoresis under nonreducing conditions reveals a single polypeptide of Mr 60 000. Macrophage tropomyosin has an isoelectric point of 4.6 and an amino acid composition similar to other tropomyosins. It contains one cysteine residue per chain. In the electron microscope, macrophage tropomyosin molecules rotary shadowed with platinum and carbon are slender, straight rods, 33 nm in length. Macrophage tropomyosin paracrystals grown in high magnesium concentrations have an axial periodicity of 34 nm. On the basis of yields from purification and from two-dimensional electrophoretic analyses of macrophage extracts, tropomyosin comprises less than 0.2% of the total macrophage protein, a molar ratio of approximately 1 tropomyosin molecule to 75 actin monomers in the cell. Macrophage tropomyosin binds to actin filaments. Macrophage, skeletal muscle, and other nonmuscle cell tropomyosins inhibit the fragmentation of actin filaments by the Ca2+-gelsolin complex. The finding implies that tropomyosin may have a role in stabilizing actin filaments in vivo.

Amino Acids↗
PubMed1983

Isolation of actin-binding protein and villin from toad oocytes.

Two actin-modulating proteins have been purified from toad oocytes. A high-molecular weight protein, similar in structure and function to macrophage actin-binding protein, accounts for the isotropic actin-crosslinking activity in oocyte homogenates. A calcium-dependent activity in toad oocyte homogenates which shortens actin filaments is accounted for by a 95,000-dalton protein which resembles villin, an actin-severing and -bundling protein of avian epithelial brush borders. In the presence of high (greater than or equal to microM) calcium, this protein shortens actin filaments in a concentration-dependent fashion and stimulates filament assembly when added to monomeric actin. In the absence of calcium the protein promotes the formation of actin filament bundles. Therefore, in the toad oocyte actin can be crosslinked into a network by actin-binding protein. Calcium regulation of the actin network may be mediated by villin. These results are different from those reported in echinoderm eggs.

Actins↗
PubMed1983

How phagocytic leukocytes move.

A regulated, coordinated movement of the cytoplasm is essential for the function of phagocytes. In these cells, as in muscle cells, the power unit for movement consists of the contractile proteins, actin and myosin, which are concentrated in the region of the cell cortex. In the peripheral cytoplasm, actin fibres may be in a fluid state or they may form a gel network by association with a homodimeric, actin-binding protein. The reversible transformation of the cytoplasm from gel to sol is mediated by a regulatory protein called gelsolin, which when activated by micromolar concentrations of Ca2+, causes shortening of actin fibres, leading to disintegration of the gel network. This gel network reforms if the Ca2+ concentration falls below the threshold value for the activation of gelsolin. Ca2+, acting via gelsolin, is a second component in this system; it controls the order of events that start on the plasma membrane of the phagocyte in response to a stimulus, and are then maintained by an appropriate reaction of the contractile unit. It is to be expected that the elucidation of the molecular mechanisms that release and regulate the movement of cytoplasm in the cell will permit an understanding of factors that interfere with leukocyte function.

Actins↗
PubMed1982

Acumentin, a protein in macrophages which caps the "pointed" end of action filaments.

Macrophages have a calcium-insensitive protein which binds to and inhibits monomer addition at the "pointed" end of actin filaments labelled with heavy meromyosin. This protein, in concert with calcium-sensitive proteins already shown to bind the "barbed" end of filaments, can lead to complete control of actin filament length in the cytoplasm. The blocking of actin filament ends in low as well as high calcium concentrations would prevent treadmilling in living cells.

Actins↗