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J H Hartwig

Publications and source records attributed to J H Hartwig.

At least 55 records · Page 3Linked to original sources
PubMed1996

Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation.

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP-280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb-IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

Actin Cytoskeleton↗
PubMed1996

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein.

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F-actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1995

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro.

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Actins↗
PubMed1995

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Androstadienes↗
PubMed1995

The ligand-induced membrane IgM association with the cytoskeletal matrix of B cells is not mediated through the Ig alpha beta heterodimer.

The B cell Ag receptor complex consists of membrane-associated Ig (mIg), Ig alpha, and Ig beta, associated molecules that have been implicated in transducing the activation signal that occurs upon receptor cross-linking. The role of the Ig alpha beta heterodimer in mediating binding to the cytoskeleton is unknown. We studied the ligand-induced association of mIgM with the cytoskeleton following receptor cross-linking in mIgM-expressing B lymphoma lines by biochemical assays, FACS analysis, and electron microscopy. Cytoskeletal association is not detected in unstimulated cells, but occurs rapidly upon anti-IgM-mediated cross-linking. Ig alpha is absent from the cytoskeleton-mIgM complex. To further analyze the possible role of Ig alpha beta in cytoskeletal binding, a surface Ig alpha beta-negative plasmacytoma line was transfected with a mutant form of mIgM (IgM-MutA). IgM-MutA is expressed on the surface despite the lack of Ig alpha beta, and the cytoskeletal binding occurred to a similar extent as in Ig-alpha-positive cell lines. In another transfectant expressing a mutated form of human mIgM (YS:VV), which does not have the capacity to bind to Ig alpha beta, the association of the receptor with the cytoskeleton appeared to be more extensive (100%) and faster than with mouse mIgM. These data indicate that Ig-associated Ig alpha beta proteins are not required for mIgM association with the cytoskeleton.

Animals↗
PubMed1995

Actin cytoskeleton. Setting the pace of cell movement.

Eukaryotic cells have many proteins that cap the barbed ends of actin filaments. Manipulation of their cellular concentration leads to changes in cell motility rates, actin dynamics and signal transduction reactions.

Actins↗
PubMed1995

Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets.

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Actins↗
PubMed1995

Actin filament cross-linking by chicken gizzard filamin is regulated by phosphorylation in vitro.

Filamin is a dimeric muscle phosphoprotein that cross-links actin filaments. We have found that purified chicken gizzard filamin is phosphorylated in vitro at serine residues by the Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Up to 0.9 mol of phosphate can be incorporated into 1 mol of filamin dimer. Phosphorylation by CaM kinase II increases filamin's critical actin filament gelling concentration and diminishes the amount of actin sedimented by filamin at low G-force. The modulation of filamin function by CaM kinase II requires ATP, Ca2+, and calmodulin, and it is abolished when CaM kinase II is inactivated with heat. Protein phosphatase 2A removed the phosphate added by CaM kinase II and restored filamin's actin filament cross-linking activity to the untreated basal level. In cosedimentation experiments, phosphorylation reduces the binding of filamin to actin filaments. The Kd for binding of filamin to actin filaments increases approximately 2-fold, from 3.2 to 6.9 microM, following CaM kinase II-mediated phosphorylation. Phosphorylation by CaM kinase II, therefore, regulates the binding of filamin to actin filaments.

Actins↗
PubMed1995

Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.

Actins↗
PubMed1995

Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin.

Gelsolin, an 82 kDa actin-binding protein, has potent actin filament-severing activity in vitro. To investigate the in vivo function of gelsolin, transgenic gelsolin-null (Gsn-) mice were generated and found to have normal embryonic development and longevity. However, platelet shape changes are decreased in Gsn- mice, causing prolonged bleeding times. Neutrophil migration in vivo into peritoneal exudates and in vitro is delayed. Gsn- dermal fibroblasts have excessive actin stress fibers and migrate more slowly than wild-type fibroblasts, but have increased contractility in vitro. These observations establish the requirement of gelsolin for rapid motile responses in cell types involved in stress responses such as hemostasis, inflammation, and wound healing. Neither gelsolin nor other proteins with similar actin filament-severing activity are expressed in early embryonic cells, indicating that this mechanism of actin filament dynamics is not essential for motility during early embryogenesis.

Actins↗
PubMed1995

Mechanism of shape change in chilled human platelets.

The so-called cold activation of platelets that precludes refrigeration of platelets for storage has long been recognized, but its mechanism has remained a mystery. Cooling of discoid resting platelets to temperatures below 15 degrees C causes shape distortions, and the chilled cells rewarmed to above 25 degrees C are spheres rather than discs. As platelet shape change responsive to receptor activation at normal temperatures requires the remodeling of an actin scaffolding (Hartwig JH, 1992, J Cell Biol 118:1421-1442), we examined the role of actin in the morphologic changes induced by cooling. The addition of actin monomers onto the fast-exchanging (barbed) ends of actin filaments accompanies the initial physiologic platelet shape changes, and a key control point in this growth is the removal of proteins (caps) from the filament ends. This uncapping of actin filament ends is mediated by polyphosphoinositide aggregates in vitro, suggesting that cold-induced phase changes in membrane lipids might uncap actin filaments and thereby account for actin assembly-mediated shape alterations during cooling. Consistent with this hypothesis, reversible inhibition of actin assembly with cytochalasin B prevented the distortions in shape, although cooled platelets had increased actin nucleation sites and became spherical. Another step in normal platelet shape changes requires the severing of actin filaments that maintain the resting platelet. The proteins that sever initially bind to the broken filament ends, and uncapping of these fragmented filaments provides numerous nucleation sites for growth of actin filaments to fill in spreading filopodia and lamellae. Actin filament fragmentation requires a rise in intracellular calcium, and we showed that chilling platelets from 37 degrees C to 4 degrees C increases free cytosolic calcium levels from 80 nmol/L to approximately 200 nmol/L in minutes, thus providing an explanation for the spherical shape of cooled, rewarmed platelets. Blocking the calcium transient with nanomolar concentrations of the permeant calcium chelators Quin-2 and Fura-2 prevented the increase in nucleation sites and the sphering, but not the other shape changes of chilled and rewarmed platelets. However, a combination of micromolar cytochalasin B and millimolar intracellular calcium chelators preserved the discoid shapes of chilled and rewarmed platelets. After removal of cytochalasin B and addition of sufficient extracellular calcium, these platelets responded with normal morphologic alterations to glass and thrombin activation.

Actin Cytoskeleton↗
PubMed1995

Interpreting photoactivated fluorescence microscopy measurements of steady-state actin dynamics.

A continuum model describing the steady-state actin dynamics of the cytoskeleton of living cells has been developed to aid in the interpretation of photoactivated fluorescence experiments. In a simplified cell geometry, the model assumes uniform concentrations of cytosolic and cytoskeletal actin throughout the cell and no net growth of either pool. The spatiotemporal evolution of the fluorescent actin population is described by a system of two coupled linear partial-differential equations. An analytical solution is found using a Fourier-Laplace transform and important limiting cases relevant to the design of experiments are discussed. The results demonstrate that, despite being a complex function of the parameters, the fluorescence decay in photoactivated fluorescence experiments has a biphasic behavior featuring a short-term decay controlled by monomer diffusion and a long-term decay governed by the monomer exchange rate between the polymerized and unpolymerized actin pools. This biphasic behavior suggests a convenient mechanism for extracting the parameters governing the fluorescence decay from data records. These parameters include the actin monomer diffusion coefficient, filament turnover rate, and ratio of polymerized to unpolymerized actin.

Actins↗
PubMed1994

Molecular cloning of chick cardiac muscle tensin. Full-length cDNA sequence, expression, and characterization.

Here we describe the molecular cloning of 7.1-kilobase cDNA encoding chick cardiac muscle tensin. It contains an open reading frame of 1,744 amino acid (aa) residues. Sequence analysis reveals that, in addition to the previously noted SH2 domain (Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q., and Chen, L. B. (1991) Science 252, 712-715), tensin contains virtually all of the known sequence (362 aa) of insertin, an actin-capping protein that allows actin monomer to be "inserted" (Schroer, E., and Wegner, A. (1985) Eur. J. Biochem. 153, 515-520). Moreover, tensin shares partial homology with actin (46.7% identity in 30 aa), beta-spectrin's actin-binding consensus (40% identity in 26 aa), BCR (40% identity in 25 aa), catenin alpha (35% identity in 45 aa), synapsin Ia (25.6% identity in 156 aa), IL-3 receptor (20.2% identity in 384 aa), and IL-2/EPO receptors (14% identity in 20 aa). Recombinant full-length tensin, tagged with an influenza-derived epitope, was over-expressed by a baculovirus system and purified to apparent homogeneity. It migrates as a 200-kDa protein in SDS-polyacrylamide gel electrophoresis, similar to the native tensin. The structure of the tensin molecule has been characterized by light scattering, electron microscopy, and gel filtration. Nine monoclonal antibodies recognizing different regions of tensin have been prepared and characterized. The epitope-tagged recombinant tensin gene was subcloned into a pRcCMV vector and transfected into NIH 3T3 cells. Immunofluorescence stainings with monoclonal antibodies specific for chick tensin (not cross-reactive with mouse tensin) showed that the expressed protein is indeed localized at focal contacts, as that of native tensin.

3T3 Cells↗
PubMed1994

Interactions of tensin with actin and identification of its three distinct actin-binding domains.

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

Actin Cytoskeleton↗
PubMed1994

Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium.

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.

Actins↗