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T P Stossel

Publications and source records attributed to T P Stossel.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of the phosphatidylinositol 4-monophosphate dissociable high-affinity profilin-actin complex.

Profilin was originally discovered in a tight complex with monomeric actin from bovine spleen, leading to its description as an actin monomer sequestering protein that maintains a pool of unpolymerized actin in cells. Subsequent purifications of profilin using different methods from diverse cells have consistently yielded preparations that affect the kinetics of actin assembly but do not efficiently maintain actin monomeric at steady state in solutions containing mM magnesium. Recent evidence that profilin inhibits phospholipase C and enhances nucleotide exchange of actin has led some to question whether profilin is ever truly an actin monomer sequestering agent. Here we report that the extraction of bovine spleen with fluoride- and pyrophosphate-containing solutions facilitates isolation of monomeric actin that is bound to profilin and does not polymerize in mM magnesium ion. The integrity of this complex depends on the presence of ATP. Phosphatidylinositol 4-monophosphate (PIP), previously shown to dissociate the low-affinity profilin-actin complex (Kd = 0.4 microM in mM Mg2+), also dissociates the high-affinity profilin-actin complex (Kd less than 0.02 microM in mM Mg2+) yielding actin that is polymerization competent and profilin that functions like profilins purified by conventional methods. Although the chemical basis of these results is not known, they indicate that profilin can tightly sequester actin monomers and support the earlier suggestion that the affinity of profilin for actin may be under metabolic control.

Actins↗
PubMed1992

Actin-binding protein requirement for cortical stability and efficient locomotion.

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Actins↗
PubMed1992

Regulation of water flow by actin-binding protein-induced actin gelatin.

Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation.

Actins↗
PubMed1991

Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.

Antibodies, Monoclonal↗
PubMed1991

Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin.

Increasing the content of the actin-binding protein gelsolin in cultured mouse fibroblasts by up to 125 percent by gene transfection proportionally enhanced the rate at which the cells migrated through porous filters toward a gradient of serum and closed a wound made on a confluent monolayer of cells in a tissue culture dish. These results provide direct evidence that gelsolin, which promotes both actin assembly and disassembly in vitro, is an important element in fibroblast locomotion and demonstrate that the manipulation of intracellular machinery can increase cell motility.

Animals↗
PubMed1990

Effect of ATP on actin filament stiffness.

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.

Actins↗
PubMed1990

Resemblance of actin-binding protein/actin gels to covalently crosslinked networks.

The maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel-sol' transformations result from the rearrangement of cortical actin-rich networks. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, alpha-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels rheologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

Actinin↗
PubMed1990

Declining American representation in leading clinical-research journals.

To determine the national origins of high-quality clinical research we looked at research articles published during the past decade in three leading general clinical-research journals, the New England Journal of Medicine, the Journal of Clinical Investigation, and the Lancet, and in a specialty journal, Blood. We found that the proportion of non-U.S. papers published annually in these journals increased between two- and almost threefold, irrespective of whether the total number published per year rose (the Journal of Clinical Investigation and Blood), fell (the Lancet), or remained constant (the New England Journal of Medicine). Most non-U.S. research published in these periodicals originated in Western Europe or Japan. The limited available data on papers sent to the journals revealed a decline in the number of U.S. papers submitted in recent years (the New England Journal of Medicine) or a slower rate of increase relative to that of non-U.S. submissions (Blood), indicating that the increase in the number of non-U.S. papers published reflects an increase in the amount of high-quality research originating abroad as compared with the amount originating in the United States. The explanation for this phenomenon is unclear, but it coincides with the slowed growth of funding from the National Institutes of Health for U.S. clinical research.

Europe↗
PubMed1990

Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin filaments. The large size of the leaves, their interruption by two hinges and flexible actin-binding site, facilitate cross-linking of widely dispersed actin filaments.

Amino Acid Sequence↗
PubMed1990

Polarization of gelsolin and actin binding protein in kidney epithelial cells.

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.

Animals↗
PubMed1989

The relationship between CR3 deficiency and neutrophil actin assembly.

Polymorphonuclear leukocytes (PMN) with a deficiency of the complement receptor type 3 (CR3) membrane glycoprotein family have impairments in the ability to adhere to surfaces as well as chemotactic and phagocytic defects, processes that require a functional contractile apparatus. PMN from the patient with neutrophil actin dysfunction (NAD) displayed similar functional characteristics to those with CR3 deficiency suggesting the two disorders may be the same disease. In order to evaluate the relationship between CR3 deficiency and actin assembly, actin filament assembly was measured in PMN from six previously reported homozygotes (two severe and four moderate CR3-deficient patients) as well as five heterozygotes for CR3 deficiency. PMN from all patients had normal unstimulated concentrations of F-actin and after exposure to the chemotactic peptide FMLP (5 x 10(-7) mol/L for 5 to 40 seconds at 25 degrees C) assembled actin normally. Pretreatment of normal PMN with concentrations of monoclonal anti-alpha CR3 antibody, capable of blocking PMN adherence, also failed to impair FMLP-induced actin filament assembly. CR3 glycoprotein expression was measured in PMNs from the mother, father, and older sister of the NAD patient (N Engl J Med 291:1093, 1974). Actin filament assembly was recently shown to be defective in PMNs from all three family members. The total concentrations of the alpha and beta CR3 subunits were below normal in PMN detergent extracts from the mother (25% of simultaneous controls) and older sister (56% of control). PMN surface expression of these two subunits was also found to be depressed (mother, 50%; older sister, 63% of control). These findings suggest these two NAD family members are heterozygote carriers for CR3 deficiency as well as NAD. Simultaneous studies of the father, however, demonstrated normal total concentrations of both the alpha and beta CR3 subunits (126% of controls) as well as normal surface expression of both subunits after phorbol myristate acetate stimulation and incubation at 37 degrees C (mean, 112% of controls) but slightly lower than normal levels after FMLP stimulation (mean, 83%). These findings indicate that CR3 deficiency generally is not associated with defective actin filament assembly and support the conclusion that NAD represents a unique kindred in which PMN actin function differs from previously reported genotypes of CR3 deficiency.

Actins↗
PubMed1989

Gelsolin-polyphosphoinositide interaction. Full expression of gelsolin-inhibiting function by polyphosphoinositides in vesicular form and inactivation by dilution, aggregation, or masking of the inositol head group.

Calcium activates, and the polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibit the mechanical severing of actin filaments by gelsolin. Previous work indicated that the physical state of the two phospholipids is important for their effects in this system. This study correlates tests of gelsolin's severing function with quasielastic light scattering measurements of the size of mixed lipid particles and shows that the previously demonstrated diminution of the maximal effect of PIP2 in micellar form by aggregation of the micelles or mixing with other phospholipids is not the result of an absolute requirement for small lipid particles, but rather the masking of critical sites by aggregation, by sequestration in multilamellar vesicles, or by dilution of the polyphosphoinositides below a critical concentration. Large unilamellar vesicles of PIP and, importantly, PIP2 at low molar ratios (less than 3%) in mixed lipid vesicles of composition similar to plasma membranes are as active as PIP2 micelles. Aggregation or masking of polyphosphoinositide head groups by neomycin or profilin, respectively, blocked inhibition of gelsolin. Experiments with bilayer-forming phospholipids or with Triton X-100 indicate that a critical number of PIP2 molecules may be required for incipient effects on a gelsolin molecule. The actin and polyphosphoinositide binding protein profilin competed with gelsolin for binding PIP2 with a stoichiometry also suggesting binding to multiple PIP2 molecules. The membrane constituents sphingosine and cholesterol blocked the effect of PIP2 on gelsolin when added alone, but did not affect PIP2 when incorporated into mixed lipid bilayers containing phosphatidylinositol. The results suggest that profilin, small changes in membrane lipid composition, and, especially, membrane PIP2 concentration could have large effects on the modulation of gelsolin function in vivo.

Animals↗
PubMed1989

Association of gelsolin with actin filaments and cell membranes of macrophages and platelets.

Recent evidence that polyphosphoinositides regulate the function of the actin-modulating protein gelsolin in vitro raises the possibility that gelsolin interacts with cell membranes. This paper reports ultrastructural immunohistochemical data revealing that gelsolin molecules localize with plasma and intracellular membranes, including rough endoplasmic reticulum, cortical vesicles and mitochondria of macrophages, and blood platelets. Anti-gelsolin gold also labeled the surface and interior of secondary lysosomes presumably representing plasma gelsolin ingested by these cells from the lung surface by endocytosis. Gelsolin molecules, visualized with colloidal gold in replicas of the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed and rapidly frozen and freeze-dried macrophages, associated with the ends of short actin filaments sitting on the cytoplasmic membrane surface. A generalized distribution of gelsolin molecules in thin sections of resting platelets rapidly became peripheral, and plasmalemma association increased following thrombin stimulation. At later times the distribution reverted to the cytoplasmic distribution of resting cells. These findings provide the first evidence for gelsolin binding to actin filament ends in cells and indicate that gelsolin functions in both cytoplasmic and membrane domains.

Actins↗
PubMed1988

Viscoelasticity of F-actin and F-actin/gelsolin complexes.

Actin is the major protein of eukaryote peripheral cytoplasm where its mechanical effects could determine cell shape and motility. The mechanical properties of purified F-actin, whether it is a viscoelastic fluid or an elastic solid, have been a subject of controversy. Mainstream polymer theory predicts that filaments as long as those found in purified F-actin are so interpenetrated as to appear immobile in measurements over a reasonable time with available instrumentation and that the fluidity of F-actin could only be manifest if the filaments were shortened. We show that the static and dynamic elastic moduli below a critical degree of shear strain are much higher than previously reported, consistent with extreme interpenetration, but that higher strain or treatment with very low concentrations of the F-actin severing protein gelsolin greatly diminish the moduli and cause F-actin to exhibit rheologic behavior expected for independent semidilute rods, and defined by the dimensions of the filaments, including shear rate independent viscosity below a critical shear rate. The findings show that shortening of actin filaments sufficiently to permit reasonable measurements brings out their viscoelastic fluid properties. Since gelsolin shortens F-actin, it is likely that the effect of high strain is also to fragment a population of long actin filaments. We confirmed recent findings that the viscosity of F-actin is inversely proportional to the shear rate, consistent with an indeterminate fluid, but found that gelsolin abolishes this unusual shear rate dependence, indicating that it results from filament disruption during the viscosity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets.

The Mr approximately 540,000 dimeric actin gelation protein, actin-binding protein (ABP), has previously been shown in human platelets to link actin to membrane glycoprotein Ib (GPIb) (Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977; Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321). We have examined further the interaction between ABP and GPIb. Platelet extracts were depleted of ABP by precipitation with anti-ABP monoclonal antibodies (mAbs); in resulting precipitates, ABP monomer is complexed with GPIb in a 5:1 molar ratio. The ABP.GPIb complex is resistant to chaotropic solvents but dissociated by the ionic detergent, sodium dodecyl sulfate. Treatment of intact platelets with the ionophore A23187 activates a Ca2+-dependent protease which cleaves the Mr approximately 270,000 ABP subunit into three fragments of Mr 190,000, 100,000, and 90,000; the latter fragment is derived from the Mr 100,000 fragment. Anti-ABP mAbs coprecipitated GPIb with the Mr 100,000 and 90,000 fragments, but not with the Mr 190,000 fragment which contains the ABP self-association site. In the reciprocal experiment, anti-GPIb antibodies co-precipitated only the Mr 100,000 and 90,000 ABP fragments. Actin also co-precipitated with the Mr 100,000 and 90,000, but not with the Mr 190,000 ABP fragment. The anti-ABP mAb that precipitated the Mr 100,000-90,000 GPIb-binding ABP fragment recognizes a trypsin cleavage fragment of ABP that binds actin filaments in vitro. These findings establish that both the GPIb-binding site and actin-binding sites are in the same region of the ABP monomer. Because of the extended bipolar conformation of the ABP molecule, the data suggest that the GPIb.actin-binding region is located remote from the self-association, or dimerization, site of the ABP subunit.

Actins↗
PubMed1988

Depression of gelsolin levels and detection of gelsolin-actin complexes in plasma of patients with acute lung injury.

Actin is the major protein of muscle and nonmuscle cells and is one of the most abundant body proteins. Physiologic or pathologic cell death may therefore result in the liberation of large amounts of this fibrous protein into the extravascular space. The potential for long actin filaments to increase plasma viscosity and change the rheology of the microvasculature are potentially obviated by the presence of 2 recently recognized plasma actin-binding proteins, vitamin-D-binding protein, and plasma gelsolin. As part of our initial evaluation of this newly recognized physiologic system in humans, we measured levels of gelsolin in plasma samples from patients with extensive lung injury. Gelsolin levels were depressed in 25 of 25 patients with the adult respiratory distress syndrome (ARDS), a disease characterized by massive cellular injury, as determined by either of 2 functional assays for gelsolin. Mean total gelsolin concentration of 20 patients with ARDS was 89.2 +/- 33 micrograms/ml (normal levels, approximately 240 micrograms/ml; p less than 0.001) and the mean free gelsolin concentration 69.6 +/- 29 micrograms/ml (normal levels, approximately 240 micrograms/ml; p less than 0.001). Gelsolin concentrations of 6 patients with bacterial pneumonias were also depressed, but to a lesser degree (mean total level, 117 +/- 21 micrograms/ml). Direct demonstration of the presence of actin in these plasmas (but not in normal plasmas) was performed by precipitating actin directly with DNase-Sepharose beads, or indirectly with antigelsolin-Sepharose beads, as confirmed with immunoblotting. Actin was found in 18 of 19 patients using DNase beads and in 7 of 19 using antigelsolin beads.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members.

A male infant with a severe neutrophil motility disorder and poorly polymerizable actin in PMN extracts was reported over a decade ago to have neutrophil actin dysfunction (NAD) (1974. N. Engl. J. Med. 291:1093-1099). Polymerized actin (F-actin) content of fixed and permeabilized intact neutrophils from the father, mother, and sister of the NAD index case have been measured using nitrobenzoxadiazole-phallacidin, a fluorescent compound which binds specifically to actin filaments. F-actin content of unstimulated PMN from all three family members was significantly lower than unstimulated control PMN (mean 23.6 +/- 0.4 SEM fluorescent units vs. 32.6 +/- 0.6 for controls). After stimulation with the chemotactic peptide FMLP, maximal F-actin content of NAD family member PMN was below that of controls (52.7 +/- 1.3 vs. 72.6 +/- 1.8). F-actin content of detergent insoluble cytoskeletons after stimulation with FMLP was also significantly lower in PMN from NAD family members as compared with controls (21 +/- 6% vs. 73 +/- 8%). PMN extracts from the father and mother, when treated with 0.6 M KCl, polymerized half as much actin as controls. Whereas diisopropylfluorophosphate treatment of normal PMN decreased actin polymerizability in cell extracts, this treatment increased the assembly of actin in parental PMN extract. Addition of purified actin to NAD extracts failed to reveal an abnormal actin polymerization inhibitory activity, and no obvious structural defect in actin purified from the father's PMNs was noted by HPLC and two dimensional thin layer chromatography of tryptic digests. The present studies of actin assembly in intact PMNs confirm that NAD is associated with a true defect in PMN actin assembly and is a genetic disorder that is recessively inherited.

Actins↗