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At least 811 records · Page 45Linked to original sources
PubMed2000

Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations.

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin beta(4). Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.

Actins↗
PubMed2000

Membrane raft association of CD47 is necessary for actin polymerization and protein kinase C theta translocation in its synergistic activation of T cells.

CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Ctheta (PKCtheta) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCtheta cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCgamma and associated Ca(2+) signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca(2+)](i) is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCtheta translocation.

Actins↗
PubMed2003

A novel mode of polymerization of alpha1-proteinase inhibitor.

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.

Amino Acid Sequence↗
PubMed2004

A neural Wiskott-Aldrich Syndrome protein-mediated pathway for localized activation of actin polymerization that is regulated by cortactin.

Activation of the epidermal growth factor (EGF) receptor can stimulate actin polymerization via the Arp2/3 complex using a number of signaling pathways, and specific stimulation conditions may control which pathways are activated. We have previously shown that localized stimulation of EGF receptor with EGF bound to beads results in localized actin polymerization and protrusion. Here we show that the actin polymerization is dependent upon activation of the Arp2/3 complex by neural Wiskott-Aldrich Syndrome protein (N-WASP) via Grb2 and Nck2. Suppression of Grb2 or Nck2 results in loss of localization of N-WASP at the activation site and reduced actin polymerization. Although cortactin has been found to synergize with N-WASP for Arp2/3-dependent actin polymerization in vitro, we find that cortactin can restrict N-WASP localization around EGF-bead-induced protrusions. In addition, cortactin-deficient cells have increased lamellipod dynamics but show reduced net translocation, suggesting that cortactin can contribute to cell polarity by controlling the extent of Arp2/3 activation by WASP family members and the stability of the F-actin network.

Actins↗
PubMed1990

Gelsolin-actin interaction and actin polymerization in human neutrophils.

The fraction of polymerized actin in human blood neutrophils increases after exposure to formyl-methionyl-leucyl-phenylalanine (fmlp), is maximal 10 s after peptide addition, and decreases after 300 s. Most of the gelsolin (85 +/- 11%) in resting ficoll-hypaque (FH)-purified neutrophils is in an EGTA resistant, 1:1 gelsolin-actin complex, and, within 5 s after 10(-7) M fmlp activation, the amount of gelsolin complexed with actin decreases to 42 +/- 12%. Reversal of gelsolin binding to actin occurs concurrently with an increase in F-actin content, and the appearance of barbed-end nucleating activity. The rate of dissociation of EGTA resistant, 1:1 gelsolin-actin complexes is more rapid in cells exposed to 10(-7) M fmlp than in cells exposed to 10(-9) M fmlp, and the extent of dissociation 10 s after activation depends upon the fmlp concentration. Furthermore, 300 s after fmlp activation when F-actin content is decreasing, gelsolin reassociates with actin as evidenced by an increase in the amount of EGTA resistant, 1:1 gelsolin-actin complex. Since fmlp induces barbed end actin polymerization in neutrophils and since in vitro the gelsolin-actin complex caps the barbed ends of actin filaments and blocks their growth, the data suggests that in FH neutrophils fmlp-induced actin polymerization could be initiated by the reversal of gelsolin binding to actin and the uncapping of actin filaments or nuclei. The data shows that formation and dissociation of gelsolin-actin complexes, together with the effects of other actin regulatory proteins, are important steps in the regulation of actin polymerization in neutrophils. Finally, finding increased amounts of gelsolin-actin complex in basal FH cells and dissociation of the complex in fmlp-activated cells suggests a mechanism by which fmlp can cause actin polymerization without an acute increase in cytosolic Ca++.

Actins↗
PubMed1977

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues.

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule-stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.

Animals↗
PubMed1978

Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.

Animals↗
PubMed1978

In vitro polymerization of microtubules from HeLa cells.

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.

Cell Fractionation↗
PubMed1982

Strongylocentrotus purpuratus spindle tubulin. I. Characteristics of its polymerization and depolymerization in vitro.

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus, repolymerized in vitro, and purified through three cycles of temperature-dependent assembly and disassembly. In addition to the tubulin, these preparations contain a protein of 80 kdaltons and a small but variable amount of actin. At 37 degrees C, the tubulin polymerizes with a critical concentration of 0.15-0.2 mg/ml into smooth-walled polymers which contain predominantly 14 protofilaments. Removal of the 80 kdalton protein and the actin by DEAE-chromatography does not change the critical concentration for polymerization. At 15 degrees C, which is within the range of physiological temperatures for S. purpuratus embryos, the spindle tubulin will self-assemble, but the rate of total polymer formation is very slow, requiring hours in the test tube. This rate can be increased by shearing the polymerizing microtubules, creating more ends for assembly, indicating that the slow rate of polymer formation is due to a slow rate of self-initiation. If spindle tubulin is polymerized at 37 degrees C and then lowered to 15 degrees C, some polymer will be retained, the percentage of which depends on the protein concentration. These results demonstrate that spindle tubulin from S. purpuratus will assemble at 37 degrees C with a low critical concentration for polymerization in the absence of detectable MAPs and will self-assemble and maintain steady state levels of polymer at physiological temperatures.

Actins↗
PubMed2002

Temperature increases associated with polymerization of irradiated PAG dosimeters.

Polyacrylamide gel (PAG) dosimeters show considerable promise as three-dimensional dosimeters for the verification of complex dose distributions associated with conformal therapy. However, the potential of PAG dosimeters has not yet been borne out in clinical practice and it is apparent that basic investigations of these dosimeters are still required. The polymerization reactions in PAG dosimeters are exothermic and the heat given off by the reactions may influence polymerization reaction kinetics. We report the results of in situ measurements of local temperature increases in irradiated PAG resulting from heat generated by the radiation-induced exothermic polymerization reactions. Temperature changes proportional to the absorbed dose were observed in the irradiated gels, reaching a maximum of 12 degrees C under high-dose conditions, depending on the thermal boundary conditions. This has practical implications, for example, using small vials of PAG to calibrate large phantoms may not be appropriate since temperature differences during irradiation between the calibration vials and phantom may alter the morphology and quantity of the polymer formed, even when irradiated to the same dose. The inhibition of radiation-induced polymerization associated with low-level oxygen contamination is manifested by a delay in the onset of temperature rise during irradiation. The observed temperature changes are used to estimate the percentage conversion of double bonds from the bis/acrylamide monomers by polymerization reactions.

Acrylic Resins↗
PubMed2002

Dual regulation of actin rearrangement through lysophosphatidic acid receptor in neuroblast cell lines: actin depolymerization by Ca(2+)-alpha-actinin and polymerization by rho.

Lysophosphatidic acid (LPA) is a potent lipid mediator with actions on many cell types. Morphological changes involving actin polymerization are mediated by at least two cognate G protein-coupled receptors, LPA(1)/EDG-2 or LPA(2)/EDG-4. Herein, we show that LPA can also induce actin depolymerization preceding actin polymerization within single TR mouse immortalized neuroblasts. Actin depolymerization resulted in immediate loss of membrane ruffling, whereas actin polymerization resulted in process retraction. Each pathway was found to be independent: depolymerization mediated by intracellular calcium mobilization, and alpha-actinin activity and polymerization mediated by the activation of the small Rho GTPase. alpha-Actinin-mediated depolymerization seems to be involved in growth cone collapse of primary neurons, indicating a physiological significance of LPA-induced actin depolymerization. Further evidence for dual regulation of actin rearrangement was found by heterologous retroviral transduction of either lpa(1) or lpa(2) in B103 cells that neither express LPA receptors nor respond to LPA, to confer both forms of LPA-induced actin rearrangements. These results suggest that diverging intracellular signals from a single type of LPA receptor could regulate actin depolymerization, as well as polymerization, within a single cell. This dual actin rearrangement may play a novel, important role in regulation of the neuronal morphology and motility during brain development.

Actinin↗
PubMed2002

Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither alpha5beta1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation.

Actins↗
PubMed2002

EB1 targets to kinetochores with attached, polymerizing microtubules.

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

Cell Nucleus↗
PubMed1987

The effect of fibrin polymerization inhibitors on quantitative measurements of plasma fibrinogen.

A number of reports have highlighted discrepancies between the DuPont aca and fibrometer-based methods for quantitating plasma fibrinogen levels. Although many authors have suggested that the presence of fibrin(ogen) degradation products are in some way responsible for the discrepancies, no direct test of this hypothesis has been carried out. In this report, the authors demonstrate that inhibitors of fibrin monomer polymerization have different effects on the aca and fibrometer assays of fibrinogen. Fibrin polymerization inhibitors allow short soluble fibrin polymers to assemble that can scatter light and are therefore detected by the aca method. However, these short polymers do not gel and thus are not detected by the fibrometer. Therefore, the fibrometer method gives lower fibrinogen values than the aca in the presence of fibrin polymerization inhibitors. The authors also assayed plasma from a patient whose fibrinogen did not polymerize normally and therefore was not measurable by the fibrometer assay. The dysfibrinogenemic plasma assayed by the aca method recorded normal fibrinogen levels. In conclusion, the DuPont aca fibrinogen assay is insensitive to alterations of fibrin polymerization whether from inhibitors or a defective fibrinogen molecule.

Blood Coagulation Tests↗
PubMed1991

A polymeric reagent for derivatization of weak nucleophiles in HPLC-UV.

Microporous and macroporous polystyrene beads are modified to contain various activated centers to which are attached a detector-sensitive label useful for analytical-scale derivatization of nucleophilic species. The effect of surface area, porosity, and percent cross-linkage of the polymeric support containing an o-nitro activated ester is investigated. The polymers are characterized by a loading determination to calculate the amount of active acylating reagent incorporated per gram of dry polymer. The kinetics of reaction with substrates of varying steric hindrance vs. solvent strength are determined with the optimized polymeric support and a second polymeric reagent is synthesized based on that support. The second polymer contains a hydroxybenzotriazole functionality. This polymer has increased electrophilicity and is used to prepare a much stronger acylating reagent than the polymeric o-nitrobenzophenol when labeled with 3,5-dinitrobenzoyl chloride. The polymeric hydroxybenzotriazole (HoBTA-DNB) is able to derivatize much weaker nucleophiles such as alcohols, thiols, and phenols at room temperature in less than 30 seconds.

Alcohols↗
PubMed1986

Age-related decline in ligand-induced actin polymerization in human leukocytes and platelets.

The change in monomeric actin was measured in human polymorphonuclear leukocytes (PMN) and platelets, using the DNase I inhibition assay, after stimulation with various agents. PMN were stimulated with the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) in 199 donors, the tumor promoter phorbol myristate acetate in 112 donors, and the hormones tri-iodothyronine and phenylephrine, in 118 donors. The donors ranged in age from 20 to 89 years. A decrease in ligand-induced actin polymerization was detected in PMN obtained from the older population, on stimulation with FMLP (p = .014) and PMA (p = .0038). In platelets stimulated with thrombin (163 donors) a significant decrease in actin polymerization also was noted in the older group (p = .017). In addition, in platelets actin polymerization was less in women compared with men (p = .042). Because actin polymerization is an event that might have an important role in cellular activation, the age-related decline in the physiological functions might be related to a decrease in ligand-induced actin polymerization.

Actins↗
PubMedDate not supplied

Measurement of cadmium-induced metallothionein in urine by ELISA and prevention of overestimation due to polymerization.

Urinary metallothionein (MT) is a biological marker of cadmium (Cd) exposure and Cd-induced renal dysfunction. The MT is prone to oxidation due to high cysteine content and forms polymers, which can result in overestimation of the protein by immunochemical methods. The objectives of the present study were to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of MT in urine and to find ways by which the protein could either be preserved in its monomeric form or converted to this form before analysis to avoid overestimation. Urine specimens analyzed were either from rats repeatedly injected with Cd or from individuals chronically exposed to cadmium through their diets. The MT in rat urine remained in the monomeric form if the urine was collected at 4 degrees C but did not if it was collected at room temperature. The MT was also polymerized if the urine was subjected to repeated freezing and thawing. Overestimation of MT in rat urine occurred (as much as 12-fold) if the MT was polymerized. Addition of 5mM mercaptoethanol to freshly collected rat urine retarded MT polymerization, and addition of 50mM mercaptoethanol converted the polymerized MT to its monomeric form. Analysis of MT in frozen human urine samples revealed that if the urines were not treated with mercaptoethanol, the estimates of MT concentration were up to 11-fold higher than in the treated samples. We conclude that the polymerization of MT in rat and human urines is a serious problem and results in overestimation of the protein by ELISA and that this problem could be overcome by the addition of mercaptoethanol to the urine samples prior to analysis.

Aged↗
PubMed2004

Sulforaphane inhibits human MCF-7 mammary cancer cell mitotic progression and tubulin polymerization.

Sulforaphane (SUL), an isothiocyanate derived from hydrolysis of glucoraphanin in broccoli and other cruciferous vegetables, was shown to induce phase II detoxification enzymes, inhibit chemically induced mammary tumors in rodents, and more recently, to induce cell cycle arrest and apoptosis in colon cancer cells. In the present study, we demonstrate that SUL also acts to inhibit proliferation of MCF-7 adenocarcinoma cells from the human breast. Treatment of synchronized MCF-7 cells with 15 micromol/L SUL resulted in significant (P < 0.05) G(2)/M cell cycle arrest (167% of control) and elevated cyclin B1 protein (175% of control) within 24 h. Moreover, 15 micromol/L SUL significantly (P < 0.05) induced phosphorylation of histone H1 (167% of control), blocked cells in early mitosis ( approximately 10-fold increase over control), and disrupted polymerization of mitotic microtubules in vivo. Subsequent exposure of purified bovine brain tubulin to relatively high doses of SUL significantly (P < 0.05) inhibited both tubulin polymerization rate (51% of control) and total tubulin polymerization (78% of control) in vitro. Additionally, polymerization of purified tubulin exposed to isothiocyanate-containing analogs of SUL was similarly inhibited. Taken together, these findings indicate that SUL has mammary cancer suppressive actions involving mitotic cell cycle arrest and suggest a mechanism linked to the disruption of normal tubulin polymerization and/or more subtle effects on microtubule dynamics.

Adenocarcinoma↗