RESUMEN
Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNF alpha). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp125FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tbeta4 overexpressing cells have approximately 1.4x more pp125FAK than the controls, and the kinase is approximately 2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp125FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp125FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp125FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.
Asunto(s)
Apoptosis/fisiología , Moléculas de Adhesión Celular/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Timosina/genética , Células 3T3/química , Células 3T3/citología , Células 3T3/enzimología , Animales , Western Blotting , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/análisis , Tamaño de la Célula/fisiología , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/fisiología , Fragmentación del ADN , Técnica del Anticuerpo Fluorescente , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica/fisiología , Ratones , Paxillin , Fosfoproteínas/análisis , Fosfoproteínas/fisiología , Proteínas Tirosina Quinasas/análisis , Timosina/análisisRESUMEN
The large G-actin pool in individual actively motile cells has been shown to be maintained primarily by the actin sequestering protein thymosin beta four (Tbeta4). It is not clear whether Tbeta4 or an isoform also plays a primary role in neural tissue containing highly motile axonal growth cones. To address this question we have made a definitive analysis of the relative contributions of all the known G-actin sequestering proteins: Tbeta4, Tbeta10, profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-actin pool in developing chick brain at embryonic days 13 and 17. From our measurements we estimate the intracellular concentration of G-actin as 30-37 microM and of Tbeta4 as 50-60 microM in an 'average' brain cell in embryonic chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to Tbeta4 is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cofilin concentrations are an order of magnitude lower than Tbeta4. Combining the contributions of Tbeta4, unphosphorylated ADF and unphosphorylated cofilin, we estimate a mean G-actin critical concentration of approximately 0.45 microM and approximately 0.2 microM, respectively, in day 13 and day 17 embryonic brain extracts, suggesting a significant developmental decrease. We conclude that (a) Tbeta4 is the major actin sequestering protein in embryonic chick brain and the only beta thymosin isoform present; (b) ADF may play a significant developmental role, as its concentration changes significantly with age; (c) the known G-actin binding proteins can adequately account for the G-actin pool in embryonic chick brain.
Asunto(s)
Actinas/metabolismo , Envejecimiento/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Proteínas Contráctiles , Proteínas del Citoesqueleto , Proteínas de Microfilamentos/metabolismo , Factores Despolimerizantes de la Actina , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Proteínas Portadoras/metabolismo , Embrión de Pollo , Destrina , Profilinas , Timosina/metabolismoRESUMEN
In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of developing muscle cells.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Timosina/fisiología , Células 3T3 , Factores Despolimerizantes de la Actina , Actinina/metabolismo , Animales , Antígenos CD/metabolismo , Northern Blotting , Proteínas Contráctiles/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Integrina alfa5 , L-Lactato Deshidrogenasa/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Miosinas/metabolismo , Profilinas , ARN Mensajero/análisis , Talina/metabolismo , Timosina/metabolismo , Factores de Tiempo , Transfección , Tropomiosina/metabolismo , Vinculina/metabolismo , Cicatrización de HeridasRESUMEN
Cultured human erythroleukemia (HEL) cells were used to study the genesis of polarity in single cells. HEL cells grow in suspension in culture medium, but attach and spread on fibronectin when treated with 10 nM phorbol myristate acetate. If the spread cells are treated with dibutyryl cyclic adenosine monophosphate, about 50% of the cells polarize and form very striking elongated processes. Time-lapse video microscopy showed that elongation develops in these cells because the anterior pole of the cell, which bears a small ruffled membrane, moves slowly (approximately 0.16 microgram/min) forward on the substratum elongating the posterior pole or tail behind it. Using indirect immunofluorescence we found that elongation of the tail correlates with the development of long microtubule bundles emanating from the centrosome, which is located posterior to the nucleus on the trailing side of the cell. Incubation with nocodazole, which inhibited development of the long microtubules and the elongation, resulted in a centrosome positioned over the nucleus in 45% of the cells and extension of the membrane ruffling to many points around the cell's periphery. Unexpectedly, time-lapse video microscopy demonstrated that the treated cultures also contained some smaller cells with very marked anterior ruffles and short tails. These cells moved rapidly about the culture dish (maximum 0.8 microgram/min; average 0.5 microgram/min). In these fast moving cells the centrosome was also located posterior to the nucleus. Several recent reports have stressed the importance of relocation of the centrosome to an anterior position in cells developing polarity after experimental wounding. Our results show that both striking polarization and rapid motility can occur without such a relocation. The polarity induced in the HEL cells correlates most clearly with the limitation of membrane ruffling to one region; this limitation is removed by microtubule disassembly. We therefore propose that localized ruffling is the critical first step in polarized motility generally, and that centrosomal position is related to other factors.
Asunto(s)
Membrana Celular/fisiología , Polaridad Celular/fisiología , Centrosoma/fisiología , Bucladesina/farmacología , Tamaño de la Célula , Humanos , Leucemia Eritroblástica Aguda , Microtúbulos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Capping of the barbed-ends of actin filaments is an important mechanism for control of the cytoskeleton. In platelets, a valuable model system, it has been thought that gelsolin was the major capping protein. We now report that platelets contain approximately 2 microM Cap Z, a calcium insensitive heterodimeric capping protein; two major and additional minor isoforms of both alpha and beta subunits are present. In lysates from resting platelets 75-80% of the Cap Z sediments with the high speed pellet, but if the platelets are activated with thrombin for 10 s, about 15% of the Cap Z leaves the pellet fraction and is found in the high speed supernatant where it is not bound to actin. This translocation of Cap Z to the supernatant is also observed when resting platelets are lysed into buffer containing 50-100 microM GTP gamma S and 10 mM EGTA. Our results suggest that release of Cap Z from some actin filaments could generate free filament barbed-ends. An increase in free barbed-ends has been reported in platelet lysates prepared shortly after thrombin activation.
Asunto(s)
Plaquetas/química , Proteínas de Microfilamentos , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Activación Plaquetaria , Actinas/metabolismo , Animales , Plaquetas/fisiología , Proteína CapZ , Fraccionamiento Químico , Pollos , Citoesqueleto/metabolismo , Electroforesis/métodos , Técnica del Anticuerpo Fluorescente Indirecta , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Immunoblotting , Proteínas Musculares/química , Proteínas Recombinantes de Fusión/inmunología , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
Thymosin beta 4 (T beta 4) binds to G-actin in vitro and inhibits actin polymerization. We studied the effects of increasing T beta 4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane-associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane-associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of T beta 4 at a concentration (approximately 28 microM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular T beta 4 concentrations were increased by three different methods: 1) microinjection of an RSV vector containing a cDNA for T beta 4; 2) transfection with the same vector; and 3) microinjection of purified T beta 4 protein. The plasmid coding for T beta 4 was microinjected into PtK2 cells together with fluorescently labeled alpha-actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha-actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2-3 days showed disassembly of stress fibers as detected by rhodamine-phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure T beta 4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, similar microinjection of pure thymosin beta 4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow regressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these T beta 4-injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process.
Asunto(s)
División Celular/fisiología , Citoesqueleto/fisiología , Timosina/fisiología , Actinina , Actinas/análisis , Animales , División Celular/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Desmina , Células Epiteliales , Interfase , Macropodidae , Microinyecciones , Mitosis/efectos de los fármacos , Mitosis/fisiología , Timosina/análisis , Timosina/genética , Timosina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
The beta thymosins are a highly conserved family of strongly polar 5 kDa polypeptides that are widely distributed among vertebrate classes; most are now known to bind to monomeric actin and inhibit its polymerization. One beta thymosin, beta four, (T beta 4) is the predominant form in mammalian cells, present at up to 0.5 mM. Many species are known to produce at least two beta thymosin isoforms, in some cases in the same cell. Their expression can be separately regulated. When present outside the cell, the N-terminal tetrapeptide of beta four appears to affect cell cycle regulation; beta thymosins or smaller fragments derived from them may have additional regulatory functions. We suggest that many developmental changes in beta thymosin levels within cells and tissues may be related to changes in G-actin pool size.
Asunto(s)
Actinas/metabolismo , Timosina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/metabolismo , Timosina/genéticaRESUMEN
Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238-246]. We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread. Spreading was approximately 85% inhibited by 100 nM staurosporine. PKC-beta was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin beta 4 decreased significantly. To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels. Staurosporine inhibited F-actin increases under all the conditions studied. Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 microM). We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F-actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms.
Asunto(s)
Actinas/efectos de los fármacos , Diglicéridos/farmacología , Leucemia Eritroblástica Aguda/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Actinas/química , Biopolímeros , Adhesión Celular/efectos de los fármacos , Activación Enzimática , Humanos , Leucemia Eritroblástica Aguda/patología , Proteína Quinasa C/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
When resting human blood platelets are stimulated with thrombin, 50 to 60% of the G-actin polymerizes to F-actin within 60 seconds. The increase in F-actin is correlated with a corresponding decrease in the complex of G-actin with T beta 4. Within 5 seconds after stimulation, nucleation sites for pyrene actin polymerization increase 1.5 times in Triton-lysed supernatants. Cytochalasin D, known to inhibit the increase in F-actin after thrombin, also inhibits the fall in T beta 4-actin complex and the increase in nucleation sites. Phosphorylation of T beta 4 could not be detected in either control or activated cells. Increased T beta 4 corresponding to that lost from the T beta 4-actin complex is present in lysates from activated platelets and retains the ability to complex with actin. The data, taken together with previous estimates for the dissociation constant of the T beta 4-actin complex, show that actin polymerization following platelet activation could be controlled primarily by the increased availability of free barbed ends of actin filaments which have a higher affinity for G-actin than does T beta 4 and suggest that the increased free T beta 4 may serve to limit the degree of polymerization.
Asunto(s)
Actinas/metabolismo , Plaquetas/química , Activación Plaquetaria , Timosina/análisis , Plaquetas/ultraestructura , Humanos , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Polímeros , Procesamiento Proteico-Postraduccional , Trombina/farmacología , Timosina/metabolismoRESUMEN
Thymosin beta 4 is a major actin monomer binding protein present at high concentration in many vertebrate cells and cell lines. The interactions of actin with thymosin beta 4, actobindin and profilin are compared. Nine beta-thymosins have been identified; six have been shown to bind to actin. Regulation of the synthesis of thymosin beta 10 and thymosin beta 4 has been found in brain development and after stimulation of several cell types, respectively. The extracellular effects of thymosin beta 4 still need clarification.
Asunto(s)
Proteínas Contráctiles , Proteínas de Microfilamentos/metabolismo , Timosina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Profilinas , Proteínas Protozoarias , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Timosina/clasificación , Timosina/genéticaRESUMEN
Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.
Asunto(s)
Actinas/metabolismo , Neutrófilos/metabolismo , Timosina/metabolismo , Actinas/aislamiento & purificación , Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito/fisiología , Humanos , Sustancias Macromoleculares , Modelos Biológicos , Neutrófilos/efectos de los fármacos , Fracciones Subcelulares/químicaRESUMEN
We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.
Asunto(s)
Actinas/metabolismo , Adenosina/metabolismo , Proteínas Contráctiles , Proteínas de Microfilamentos/metabolismo , Timosina/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Unión Competitiva , Plaquetas/metabolismo , Humanos , Fosfatidilinositoles/metabolismo , Profilinas , Timosina/metabolismoRESUMEN
Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Músculos/metabolismo , Timosina/análogos & derivados , Actinas/sangre , Actinas/aislamiento & purificación , Animales , Cromatografía en Gel , Humanos , Cinética , Sustancias Macromoleculares , Conejos , Timosina/metabolismo , Timosina/farmacologíaRESUMEN
It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15-E21) reaching lengths of 20 microns or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F-actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F-actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 +/- 0.5 micrograms/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 micrograms/ml (13 microM). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in beta actin mRNA and a four- to fivefold increase in message for gamma actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA.
Asunto(s)
Actinas/metabolismo , Epitelio Pigmentado Ocular/embriología , ARN Mensajero/metabolismo , Actinas/genética , Animales , Northern Blotting , Células Cultivadas , Embrión de Pollo , Desoxirribonucleasas/metabolismo , Microscopía Fluorescente , Microvellosidades/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , ARN Mensajero/genética , Vimentina/análisisRESUMEN
To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin-stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Plaquetas/ultraestructura , Gránulos Citoplasmáticos/ultraestructura , Citoesqueleto de Actina/química , Actinas/análisis , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Plaquetas/química , Proteínas Sanguíneas/análisis , Calcio/análisis , Fraccionamiento Celular , Proteínas del Citoesqueleto/análisis , Ácido Egtácico , Éteres Cíclicos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica , Miosinas/análisis , Miosinas/metabolismo , Ácido Ocadaico , Permeabilidad , Saponinas/farmacología , TalinaRESUMEN
At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/química , Timosina/análogos & derivados , Secuencia de Aminoácidos , Plaquetas/química , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Timosina/química , Timosina/metabolismoRESUMEN
To investigate the function of vinculin in blood platelets, we studied its localization in relation to other cytoskeletal proteins as well as its state of phosphorylation in platelets allowed to spread on fibrinogen-coated surfaces. By 5 minutes after loading the platelets onto the surfaces the 47 and 20 kDa polypeptides became phosphorylated, indicating activation. By 30 minutes, platelets formed small, typical bundles of fibers which stained brilliantly with rhodamine phalloidin. Myosin and tropomyosin, detected with specific antibodies, were localized in periodic arrays along these bundles. By indirect immunofluorescence, a discrete patch of vinculin was observed at each end of every actin-containing bundle. Vinculin phosphorylation was not detected in immunoprecipitates protected against phosphatases. Interference reflection images showed that regions of close binding to the substratum (adhesion plaques) closely matched the vinculin staining sites. Talin appeared diffusely localized. It could be shown to be present in the plaques when platelets were stabilized with ZnCl2 by the method of Geiger and then sonicated to remove some of the surface membrane. Localizations of vinculin and myosin were unaltered by this treatment. Talin phosphorylation or proteolysis could not account for vinculin translocation. We conclude that platelets, in response to an appropriate physiological surface, form typical actin bundles with vinculin at the termination of each bundle, in close relation to adhesion plaques. The signal for this translocation does not appear to depend on phosphorylation of vinculin or on phosphorylation or proteolysis of talin. Our findings support the conclusion that in platelets, as in nucleated cells, vinculin serves as at least part of the connection between bundled actin fibers and the extracellular matrix. Such a connection seems required for platelets' known ability to exert tension on surfaces.
Asunto(s)
Plaquetas/ultraestructura , Vinculina/fisiología , Compuestos de Zinc , Actinas/análisis , Plaquetas/química , Plaquetas/efectos de los fármacos , Adhesión Celular , Cloruros/farmacología , Citoesqueleto/ultraestructura , Fibrinógeno , Técnica del Anticuerpo Fluorescente , Humanos , Miosinas/análisis , Fosforilación , Activación Plaquetaria , Procesamiento Proteico-Postraduccional , Talina/análisis , Tropomiosina/análisis , Vinculina/metabolismo , Zinc/farmacologíaRESUMEN
Resting human platelets contain approximately 0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.
Asunto(s)
Actinas/sangre , Plaquetas/metabolismo , Proteínas de Microfilamentos/sangre , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Cinética , Sustancias Macromoleculares , Proteínas de Microfilamentos/aislamiento & purificación , Peso Molecular , Músculos/metabolismo , Miosinas/metabolismo , ConejosRESUMEN
Sequestration of calcium into an intracellular storage site is an important mechanism in helping to maintain a low cytoplasmic Ca2+ level in many cells. In platelets, increasing cytoplasmic cAMP lowers the free calcium level in correlation with the phosphorylation of a 22 kD protein. This protein has been thought to enhance uptake of calcium into a platelet membrane bound storage site by activating a calcium-ATPase activity by analogy with phospholamban in cardiac muscle. The evidence for an analogue of phospholamban in platelets is unclear. A pathway involving cAMP dependent kinase also seems unlikely to account for the transience of the calcium signal following agonists in platelets, some of which inhibit the cAMP dependent kinase. Here we discuss the issue of whether activation of protein kinase C, which follows agonist action, leads to enhanced calcium sequestration in platelets and if so, what indications there are for a mechanism. The evidence from our experiments with phorbol myristate acetate treated platelets shows that such an enhancement can be produced by activating protein kinase C. Phosphorylation studies suggest the involvement of a polypeptide or polypeptides distinct from the 22 kD polypeptide. Further work to test this idea is necessary. A brief overview of research on the role of phosphoproteins in calcium regulation in platelets and comparison with their role in cardiac muscle is also presented.