RESUMEN
Context: College soccer players suffer from hamstring injuries due to inflexibility and repetitive motions involving intense hamstring lengthening and contraction during sport. Although it is a popular intervention for muscular injury, there exists limited evidence of the effects of therapeutic cupping on hamstring flexibility. Objective: To determine the effect of cupping therapy on hamstring flexibility in college soccer players. Design: Cohort design. Setting: Athletic training clinic. Patients: A total of 25, asymptomatic, National Collegiate Athletic Association Division III soccer players (10 males and 15 females; age = 19.4 [1.30] y, height = 175.1 [8.2] cm, and mass = 69.5 [6.6] kg). Intervention(s): A 7-minute therapeutic cupping treatment was delivered to the treatment group. Four 2-in cups were fixed atop trigger point locations within the hamstring muscle bellies of participants' dominant legs. Control group participants received no intervention between pretest and posttest measurements. Main Outcome Measures: Pretest and posttest measurements of hamstring flexibility, using a passive straight leg raise, were performed on both groups. Passive straight leg raise measurements were conducted by blinded examiners using a digital inclinometer. An independent samples t test was used to analyze changes in hamstring flexibility from pretreatment to posttreatment with P values set a priori at .05. Results: An independent samples t test demonstrated no significant difference in change in hamstring flexibility between participants in the treatment group and those in the control group (t23 = -.961, P = .35). Conclusions: The findings of this study demonstrated no statistically significant changes in hamstring flexibility following a cupping treatment.
Asunto(s)
Terapias Complementarias , Elasticidad , Músculos Isquiosurales/fisiología , Traumatismos de la Pierna/prevención & control , Adolescente , Femenino , Humanos , Masculino , Músculo Esquelético/lesiones , Fútbol , Adulto JovenRESUMEN
DIF-1 (differentiation-inducing factor1) is a polyketide produced by Dictyostelium prespore cells which induces initially uncommitted cells to differentiate as prestalk cells. Exposure of cells to DIF-1 causes transitory hypo-phosphorylation of seven serine residues in YelA, a protein with a region of strong homology to the MIF4G domain of the eukaryotic initiation factor eIF4G. Based upon its domain architecture, which in one important aspect closely resembles that of Death-Associated Protein 5 (DAP5), we predict a role in stimulating internal ribosome entry-driven mRNA translation. The two paradigmatic DIF-1 inducible genes are ecmA (extracellular matrixA) and ecmB. In support of a YelA function in DIF-1 signaling, a YelA null strain showed greatly increased expression of ecmA and ecmB in response to DIF-1. Also, during normal development in the null strain, expression of the two genes is accelerated. This is particularly evident for ecmB, a marker of stalk tube and supporting structure differentiation. Mutants in DIF-1 bio-synthesis or signaling display a rudimentary or no basal disc and, conversely, YelA null mutants produce fruiting bodies with a highly enlarged basal disc that ectopically expresses a stalk tube-specific marker. Thus YelA acts as an antagonist of DIF-1 signaling, with a consequent effect on cell type proportioning and it is predicted to act as a translational regulator.
Asunto(s)
Dictyostelium/metabolismo , Hexanonas/farmacología , Proteínas Protozoarias/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Cuerpos Fructíferos de los Hongos/efectos de los fármacos , Cuerpos Fructíferos de los Hongos/genética , Cuerpos Fructíferos de los Hongos/metabolismo , Microorganismos Modificados Genéticamente , Fosforilación/efectos de los fármacos , Proteínas Protozoarias/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1-controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca(2+)/calmodulin-dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555.
Asunto(s)
Dictyostelium/metabolismo , Hexanonas/metabolismo , Hidrocarburos Clorados/metabolismo , Policétidos/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Calcineurina/química , Quimiotaxis , AMP Cíclico/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Fosforilación , Transducción de SeñalRESUMEN
When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase-like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.
Asunto(s)
Dictyostelium/enzimología , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/fisiología , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Dominios Homologos srcRESUMEN
Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca(2+)-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3(-), phg2(-), and pyk3(-)/phg2(-) cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca(2+) liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3(-) and phg2(-) cells and even further decreased in pyk3(-)/phg2(-) cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2(-) cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues.
Asunto(s)
Dictyostelium/enzimología , Presión Osmótica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción STAT/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Dictyostelium/citología , Dictyostelium/metabolismo , Mutación , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Transducción de SeñalRESUMEN
INTRODUCTION/BACKGROUND: Optimal baseball throwing mechanics require a significant contribution of thoracolumbar motion, particularly in the sagittal and transverse planes. This motion is key for proper transmission of forces from the lower to upper extremity, thereby minimizing a throwing athlete's risk of injury and maximizing athletic performance. PURPOSE: To define the active-assisted thoracolumbar ROM of both baseball pitchers and position players and to compare these motions both within and between groups. METHODS: Fifty-six asymptomatic, collegiate and minor league baseball pitchers and 42 position players volunteered to participate. Active-assisted thoracolumbar flexion, extension, and bilateral rotation ROM, were measured in a standing position, using two bubble inclinometers. Two-tailed t tests were used to determine differences in ROM between and within the pitchers and position players. RESULTS: The pitchers had significantly more rotation to the non-throwing arm side as compared to the position players (p = .007, effect size = .61). The pitchers also had more rotation to the non-throwing arm side as compared to their throwing side (p = .006, effect size = .47). There were no other significant differences between the pitchers and the position players (p > .53). Furthermore, the position players did not have a side-to-side rotation difference (p = .99). CONCLUSIONS: Pitchers have a greater amount of rotation ROM towards the non-throwing arm side as compared to position players. Pitchers also have a greater amount of rotation ROM to the non-throwing arm side as compared to their throwing side rotation. Because pitchers often present with posterior shoulder tightness and subsequent altered shoulder horizontal adduction and internal rotation ROM, the increase in non-throwing side rotation ROM may occur in response to these adaptations. More specifically, this increase in non-throwing side trunk rotation ROM may allow such athletes to bring the arm across the body during the follow-through phase of the throwing motion despite posterior shoulder tightness. However, future research is necessary to investigate this relationship. Based on these results, clinicians should consider these thoracolumbar ROM adaptations in the prevention, evaluation, and treatment of baseball players. LEVEL OF EVIDENCE: 2b.
RESUMEN
BACKGROUND: Cell behaviour is tightly determined by sensing and integration of extracellular changes through membrane detectors such as receptors and transporters and activation of downstream signalling cascades. Arrestin proteins act as scaffolds at the plasma membrane and along the endocytic pathway, where they regulate the activity and the fate of some of these detectors. Members of the arrestin clan are widely present from unicellular to metazoa, with roles in signal transduction and metabolism. As a soil amoeba, Dictyostelium is frequently confronted with environmental changes likely to compromise survival. Here, we investigated whether the recently described arrestin-related protein AdcA is part of the cell response to stresses. RESULTS: Our data provide evidence that AdcA responds to a variety of stresses including hyperosmolarity by a transient phosphorylation. Analysis in different mutant backgrounds revealed that AdcA phosphorylation involves pathways other than the DokA and cGMP-dependent osmostress pathways, respectively known to regulate PKA and STATc, key actors in the cellular response to conditions of hyperosmolarity. Interestingly, however, both AdcA and STATc are sensitive to changes in the F-actin polymerization status, suggesting a common primary sensor/trigger and linking the stress-sensitive kinase responsive for AdcA phosphorylation to the actin cytoskeleton. We also show that STATc-dependent transcriptional activity is involved for the timely dephosphorylation of AdcA in cells under stress. CONCLUSION: Under osmotic stress, AdcA undergoes a phosphorylation-dephosphorylation cycle involving a stress-sensitive kinase and the transcription regulator STATc. This transient post-transcriptional modification may allow a regulation of AdcA function possibly to optimize the cellular stress response.
Asunto(s)
Arrestinas/fisiología , Proteínas Protozoarias/fisiología , Factores de Transcripción STAT/fisiología , Estrés Fisiológico/fisiología , Dictyostelium/fisiología , Presión Osmótica , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
MrfA, a transcription factor that regulates Dictyostelium prestalk cell differentiation, is an orthologue of the metazoan myelin gene regulatory factor (MRF) proteins. We show that the MRFs contain a predicted transmembrane domain, suggesting that they are synthesised as membrane-tethered proteins that are then proteolytically released. We confirm this for MrfA but report a radically different mode of processing from that of paradigmatic tethered transcriptional regulators, which are cleaved within the transmembrane domain by a dedicated protease. Instead, an auto-proteolytic cleavage mechanism, previously only described for the intramolecular chaperone domains of bacteriophage tail-spike proteins, processes MrfA and, by implication, the metazoan MRF proteins. We also present evidence that the auto-proteolysis of MrfA occurs rapidly and constitutively in the ER and that its specific role in prestalk cell differentiation is conferred by the regulated nuclear translocation of the liberated fragment.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Proteínas de la Membrana/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Proteínas de la Cola de los Virus/genética , Secuencia de Aminoácidos , Bacteriófagos/genética , Diferenciación Celular , Dictyostelium/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicósido Hidrolasas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Proteolisis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de la Cola de los Virus/metabolismoRESUMEN
DIF-1, a chlorinated hexaphenone produced by developing Dictyostelium cells, induces prestalk differentiation. DimB is a bZIP transcription factor that accumulates in the nucleus upon exposure to DIF-1, where it directly activates transcription of DIF-responsive genes. The signaling steps upstream of DimB and downstream of DIF-1 are entirely unknown. Analysis by mass spectrometry shows that incubation with DIF-1 rapidly stimulates phosphorylation at several sites in DimB. We characterize the most highly responsive site, S590, which is located very close to the C terminus. A point mutation in this site, S590A, does not inhibit DimB nuclear accumulation in response to DIF. However, this seems likely to reflect functional redundancy with other sites; because a panel of chemical variants on the structure of DIF-1 show a correlation between their potencies as inducers of DimB nuclear accumulation and their potencies as inducers of phosphorylation at S590. Furthermore, the S590A mutant is fully active in mutant rescue of a dimB null strain, arguing against an alternative role in transcriptional activation of target genes. We conclude that i) DIF-1 directs phosphorylation at S590, ii) although it is not essential for nuclear accumulation in response to DIF-1 correlative evidence, based upon a panel of DIF-1 related molecules, suggests that this modification may play a redundant role in the process. iii) We also present evidence that the kinase activity, which phosphorylates S590, is non-nuclear and that this signalling pathway is, in part at least, independent of the DIF-regulated STATc activation pathway.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Dictyostelium/citología , Hexanonas/farmacología , Proteínas Protozoarias/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Western Blotting , Núcleo Celular/metabolismo , Dictyostelium/química , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Mutación Puntual , Proteínas Protozoarias/genética , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Serina/genética , Serina/metabolismoRESUMEN
PURPOSE/BACKGROUND: To compare the acute effects of two passive stretches on pectoralis minor length and scapular kinematics among a group of collegiate swimmers. METHODS: The study was a descriptive design with repeated measures. All procedures were conducted in a biomechanics laboratory and collegiate swimming facility. Fifty asymptomatic shoulders from 29 NCAA swimmers were used (15 control shoulders, 17 focused stretch shoulders, 18 gross stretch shoulders). Pre- and post-test linear pectoralis minor length, as well as scapular kinematics (upward/downward rotation, external/internal rotation, anterior/posterior tilt) were measured as dependent variables. Pectoralis minor length was measured using a standard tape measure and three-dimensional scapular kinematics were measured using an electromagnetic capture system. RESULTS: The gross stretch shoulders had a significant increase in pectoralis minor length compared to the control shoulders (P=.007). There were no other significant changes in length for either the focused stretch or control shoulders (P>.07). No statistically significant (P>.08) differences for all three scapular kinematic variables were found among any of the three groups (P>.08). CONCLUSIONS: Our results revealed no acute improvements of scapular upward rotation, external rotation, or posterior tilt after the application of either passive stretch maneuver to the pectoralis minor muscle. LEVEL OF EVIDENCE: 2b.
RESUMEN
OBJECTIVES: To determine the strength of the relationship between latissimus dorsi stiffness and altered scapular kinematics among swimmers. DESIGN: Cross sectional. SETTING: Laboratory. PARTICIPANTS: Nineteen NCAA Division III swimmers (7 male, 12 female) (age = 18.8 ± 0.9 years, height = 174.7 ± 8.9 cm, mass = 71.6 ± 11.9 kg) volunteered to participate. Subjects had no recent history of upper extremity pathology or any previous surgery. INTERVENTIONS: We measured latissimus dorsi stiffness of the dominant arm while in a lengthened position with a myotonometer. We used an electromagnetic tracking device with specialized software to measure scapular kinematics at humeral elevation angles of 30°, 60°, 90°, and 110° within the scapular plane. MAIN OUTCOME MEASURES: Latissimus dorsi stiffness and scapular upward/downward rotation, internal/external rotation, and anterior/posterior tilt. RESULTS: Latissimus dorsi stiffness showed moderate-to-good relationships with increased scapular upward rotation (r > -0.63, P < 0.002) and posterior tilt (r > -0.62, P < 0.004) at all four angles of humeral elevation. Increased latissimus dorsi stiffness also showed moderate-to-good relationships with decreased scapular internal rotation at humeral elevation angles of 60° (r = 0.47, P = 0.03) and 90° (r = 0.54, P = 0.01). CONCLUSIONS: Our results suggest there are several moderate-to-good relationships between increased latissimus dorsi stiffness in swimmers and altered scapular upward rotation, internal rotation, and posterior tilt at various angles of humeral elevation. If latissimus dorsi stiffness is not addressed subsequent scapular alterations, which have been associated with shoulder dysfunction, may occur.
Asunto(s)
Ejercicio Físico/fisiología , Fatiga Muscular/fisiología , Músculo Esquelético/fisiología , Escápula/fisiología , Natación/fisiología , Adolescente , Fenómenos Biomecánicos , Estudios Transversales , Femenino , Humanos , MasculinoRESUMEN
The prestalk region of the Dictyostelium slug is comprised of an anterior population of pstA cells and a posterior population of pstO cells. They are distinguished by their ability to utilize different parts of the promoter of the ecmA gene. We identify, by mutational analysis and DNA transformation, CA-rich sequence elements within the ecmA promoter that are essential for pstA-specific expression and sufficient to direct pstA-specific expression when multimerised. The CA-rich region was used in affinity chromatography with nuclear extracts and bound proteins were identified by mass spectrometry. The CA-rich elements purify MrfA, a protein with extensive sequence similarity to animal Myelin-gene Regulatory Factor (MRF)-like proteins. The MRF-like proteins and MrfA also display more spatially limited but significant sequence similarity with the DNA binding domain of the yeast Ndt80 sporulation-specific transcription factor. Furthermore, the ecmA CA-rich elements show sequence similarity to the core consensus Ndt80 binding site (the MSE) and point mutation of highly conserved arginine residues in MrfA, that in Ndt80 make critical contacts with the MSE, ablate binding of MrfA to its sites within the ecmA promoter. MrfA null strains are delayed in multicellular development and highly defective in pstA-specific gene expression. These results provide a first insight into the intracellular signaling pathway that directs pstA differentiation and identify a non-metazoan orthologue of a family of molecularly uncharacterised transcription factors.
Asunto(s)
Diferenciación Celular , Dictyostelium/crecimiento & desarrollo , Vaina de Mielina/genética , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Cartilla de ADN/química , Dictyostelium/genética , Dictyostelium/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.
Asunto(s)
Dictyostelium/metabolismo , Quinasa 2 de Adhesión Focal/genética , Hexanonas/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/fisiología , Dominios Homologos src/genética , Evolución Molecular , Quinasa 2 de Adhesión Focal/metabolismo , Glutatión Transferasa/metabolismo , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Tirosina Quinasas/metabolismo , Tirosina/químicaRESUMEN
The Dictyostelium transcription factor STATc is tyrosine phosphorylated and accumulates in the nucleus when cells are exposed either to hyper-osmotic stress or to the prestalk-inducing polyketide DIF-1. In the case of stress STAT activation is mediated by regulated dephosphorylation; whereby two serine residues on PTP3, the tyrosine phosphatase that de-activates STATc, become phosphorylated after exposure to stress so inhibiting enzymatic activity. We now show that the more highly regulated of the two PTP3 serine residues, S747, is also phosphorylated in response to DIF-1, suggesting a common activation mechanism. Hyper-osmotic stress causes a re-distribution of F-actin to the cortex, cell rounding and shrinkage and we show that DIF-1 induces a similar but transient F-actin re-distribution and rounding response. We also find that two mechanistically distinct inhibitors of actin polymerization, latrunculin A and cytochalasin A induce phosphorylation at S747 of PTP3 and activate STATc. We suggest that PTP3 phosphorylation, and consequent STATc activation, are regulated by changes in F-actin polymerization status during stress and DIF-induced cytoskeletal remodelling.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Actinas/metabolismo , Calcineurina/metabolismo , Dictyostelium/citología , Hexanonas/metabolismo , Fosforilación/fisiologíaRESUMEN
In response to the signaling polyketide DIF-1 DimB directly activates transcription of the ecmB gene in pstB cells; a subset of the prestalk cells that are the precursors of the basal disc. We show that the promoter of pspA, a prespore-specific gene, also contains a DimB binding site. Mutation of this site causes ectopic expression in the prestalk region and ChIP analysis shows that DIF-1 induces binding of DimB to the pspA promoter. DIF-1 represses pspA gene expression in a suspension cell assay but this repression is abrogated in a dimB null strain. These results suggest a coupled control mechanism, whereby the same DIF-DimB signaling pathway that directly activates ecmB gene expression directly represses pspA gene expression.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular , Dictyostelium/citología , Dictyostelium/genética , Proteínas Protozoarias/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/aislamiento & purificación , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Cromatografía de Afinidad , Dictyostelium/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Hexanonas/farmacología , Hidrocarburos Clorados/farmacología , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Esporas Protozoarias/citología , Esporas Protozoarias/efectos de los fármacos , Esporas Protozoarias/genética , Transcripción Genética/efectos de los fármacosRESUMEN
SmdA is a Dictyostelium orthologue of the SET/MYND chromatin re-modelling proteins. In developing structures derived from a null mutant for smdA (a smdA- strain), prestalk patterning is normal, but using a prespore lacZ reporter fusion, there is ectopic accumulation of beta-galactosidase in the prestalk region. As wild type slugs migrate, there is continual forward movement and re-differentiation of prespore cells into prestalk cells. Thus, a potential explanation for the ectopic reporter localization in smdA null prestalk cells is an increased rate of re-differentiation and anterior movement of prespore cells. In support of this notion, analysis of an unstable lacZ reporter, driven by the prespore promoter, reveals a normal staining pattern in the smdA- strain. We suggest that one or more genes regulated by SmdA acts to repress prespore re-specification.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Dictyostelium/genética , Proteínas Protozoarias/genética , Factores de Transcripción/genética , Diferenciación Celular/genética , Dictyostelium/citología , Dictyostelium/fisiología , Operón Lac , Modelos Biológicos , Nucleoproteínas/genética , Regiones Promotoras Genéticas , Proteínas Protozoarias/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Esporas/crecimiento & desarrollo , beta-Galactosidasa/genéticaRESUMEN
Exposure of monolayer Dictyostelium cells to the signalling polyketide DIF-1 causes DimB, a bZIPtranscription factor, to accumulate in the nucleus where it induces prestalk gene expression. Here we analyse DimB signalling during normal development. In slugs DimB is specifically nuclear enriched in the pstB cells; a cluster of vital dye-staining cells located on the ventral surface of the posterior, prespore region. PstB cells move at culmination, to form the lower cup and the outer basal disc of the fruiting body, and DimB retains a high nuclear concentration in both these tissues. In a dimB null (dimB-) strain there are very few pstB or lower cup cells, as detected by neutral red staining, and it is known that the outer basal disc is absent or much reduced. In the dimB- strain ecmB, a marker of pstB differentiation, is not DIF inducible. Furthermore, ChIP analysis shows that DimB binds to the ecmB promoter in DIF-induced cells. These results suggest that the differentiation of pstB cells is caused by a high perceived level of DIF-1 signalling, leading to nuclear localization of DimB and direct activation of cell type-specific gene expression.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Dictyostelium/metabolismo , Hexanonas/farmacología , Proteínas Protozoarias/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Dictyostelium/citología , Dictyostelium/fisiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Movimiento/fisiología , Mutación , Unión Proteica , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein-protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Hexanonas/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , ADN Protozoario/genética , Oscuridad , Dictyostelium/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Luz , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Transducción de Señal , Dominios Homologos srcRESUMEN
cotC requires the transcription factor CudA for its expression in the posterior, prespore cells of the slug, while the expL7 gene requires CudA for its expression in the anterior, tip-organiser region. In order to identify additional transcription factors that might mediate tip-organiser specific expression, we performed affinity chromatography on slug nuclear extracts. The affinity matrix bore cap-site distal sequences from region A of the expL7 promoter; an essential region located upstream of the CudA binding domain. One of the proteins purified was G-box binding factor (GBF), a zinc finger transcription factor which binds to G-rich elements, known as G boxes, that are present in the promoters of many developmental genes, including cotC. Previous work identified an essential sequence motif within region A and we show that this element is a G box, that binds recombinant GBF. Moreover, a G box from within the cotC promoter can substitute for region A of expL7 in directing tip-organiser specific expression of expL7. Thus the same two transcription factors, CudA and GBF, seem to co-operate to direct both tip-organiser and prespore gene expression. How then is specificity achieved? Replacing a CudA binding region in the cotC promoter with the CudA binding domain from expL7 strongly represses cotC promoter activity. Hence we suggest that differences in the topology of the multiple CudA half- sites contained within the two different CudA binding regions, coupled with differences in the signalling environment between tip-organiser cells and prespore cells, ensure correct expL7 expression.
Asunto(s)
Dictyostelium/genética , Regulación de la Expresión Génica , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Dictyostelium/citología , Dictyostelium/metabolismo , Factores de Unión a la G-Box/metabolismo , Secuencia Rica en GC/genética , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Protozoarias/genética , Factores de Transcripción/genéticaRESUMEN
Any established or aspiring model organism must justify itself using two criteria: does the model organism offer experimental advantages not offered by competing systems? And will any discoveries made using the model be of wider relevance? This review addresses these issues for the social amoeba Dictyostelium and highlights some of the organisms more recent applications. These cover a remarkably wide gamut, ranging from sociobiological to medical research with much else in between.