RESUMEN
CONTEXT: Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. OBJECTIVES: This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. MATERIALS AND METHODS: HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. RESULTS: The IC50 of arecoline was approximately 50 µg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-ß receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. DISCUSSION AND CONCLUSIONS: Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-ß/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.
Asunto(s)
Arecolina/toxicidad , Fibrosis de la Submucosa Bucal/inducido químicamente , Proteínas Smad/fisiología , Factor de Crecimiento Transformador beta/fisiología , Tropomiosina/genética , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Células HaCaT , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tropomiosina/fisiología , Regulación hacia ArribaRESUMEN
Tropomyosins regulate the dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of the fluorescently labeled tropomyosin isoform Tpm1.8 to unlabeled actin filaments in real time. This approach, in conjunction with mathematical modeling, enabled us to quantify the nucleation, assembly, and disassembly kinetics of Tpm1.8 on single filaments and at the single-molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilized by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented toward the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.
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Citoesqueleto de Actina/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Cinética , Microfluídica/métodos , Microscopía Fluorescente/métodos , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/metabolismo , Tropomiosina/fisiologíaRESUMEN
Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The α- (Tpm 1.1) and γ- (Tpm 3.12) Tpm isoforms are expressed in fast and slow human skeletal muscles, respectively, while ß-Tpm (Tpm 2.2) is expressed in both muscle types. This results in the formation of Tpm αα- and γγ-homodimers as well as αß- and γß-heterodimers. The properties of αα-homodimer are well studied, whereas very little is known about the functional properties of γγ-homodimer and γß-heterodimer. We investigated interaction characteristics of Tpm γγ-homodimer and γß-heterodimer with actin filaments and Ca2+-regulation of actin-myosin interaction on myosin from fast and slow skeletal muscles. The results showed that complexes formed by γγ-Tpm and γß-Tpm with F-actin are more stable than those with αα-Tpm and αß-Tpm. The maximum sliding speed of regulated thin filaments with either γγ-Tpm or γß-Tpm moving over skeletal myosin was significantly less than that of the filaments with αα-Tpm or αß-Tpm. The results indicate that isoforms of Tpm along with isoforms of myosin determine of functional properties of skeletal muscles and support an idea on the combined expression of myosin and Tpm isoforms.
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Músculo Esquelético/metabolismo , Tropomiosina , Calcio/fisiología , Humanos , Contracción Muscular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Multimerización de Proteína , Tropomiosina/química , Tropomiosina/fisiologíaRESUMEN
Platelets are implicated in the pathophysiology of breast and other cancers through their role in exchanging biomolecules with tumor cells in the tumor microenvironment. Such exchange results in tumor-educated platelets with altered RNA expression profiles. Multiple lines of evidence indicate that platelet RNA profiles may be suitable as diagnostic biomarkers for cancer-related biological processes. In this study, we characterized the gene expression signatures of platelets in breast cancer (BC) by high-throughput sequencing and quantitative real-time RT-PCR. Our results indicate that the expression of TPM3 (tropomyosin 3) mRNA is significantly elevated in platelets from patients with BC compared with age-matched healthy control subjects. Furthermore, up-regulation of TPM3 mRNA in platelets was found to be significantly correlated with metastasis in patients with BC. Finally, we report that platelet TPM3 mRNA is delivered into BC cells through microvesicles and leads to enhanced migrative phenotype of BC cells. In summary, our findings suggest that the transfer of platelet TPM3 mRNA into cancer cells via microvesicles promotes cancer cell migration, and thus platelet-derived TPM3 mRNA may be a suitable biomarker for early diagnosis of metastatic BC.
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Neoplasias de la Mama/genética , Micropartículas Derivadas de Células/genética , Tropomiosina/metabolismo , Adulto , Anciano , Biomarcadores de Tumor , Plaquetas/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/fisiopatología , Línea Celular Tumoral , Movimiento Celular , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Procesos Neoplásicos , ARN Mensajero/genética , Transcriptoma/genética , Tropomiosina/fisiología , Microambiente Tumoral/fisiologíaRESUMEN
Substitution of Ala for Thr residue in 155th position in γ-tropomyosin (Tpm3.12) is associated with muscle weakness. To understand the mechanisms of this defect, we studied the Ca2+-sensitivity of thin filaments in solution and multistep changes in mobility and spatial arrangement of actin, Tpm, and myosin heads during the ATPase cycle in reconstituted muscle fibres, using the polarized fluorescence microscopy. It was shown that the Ala155Thr (A155T) mutation increased the Ca2+-sensitivity of the thin filaments in solution. In the absence of the myosin heads in the muscle fibres, the mutation did not alter the ability of troponin to switch the thin filaments on and off at high and low Ca2+, respectively. However, upon the binding of myosin heads to the thin filaments at low Ca2+, the mutant Tpm was found to be markedly closer to the open position, than the wild-type Tpm. In the presence of the mutant Tpm, switching on of actin monomers and formation of the strong-binding state of the myosin heads were observed at low Ca2+, which indicated a higher myofilament Ca2+-sensitivity. The mutation decreased the amount of myosin heads bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation. It is suggested that direct binding of myosin to Tpm may be one оf the reasons for muscle weakness associated with the A155T mutation. The use of reagents that decrease the Ca2+-sensitivity of the troponin complex may not be adequate to restore muscle function in patients with the A155T mutation.
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Calcio/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/fisiopatología , Tropomiosina/genética , Tropomiosina/fisiología , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Animales , Polarización de Fluorescencia , Humanos , Técnicas In Vitro , Masculino , Debilidad Muscular/etiología , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Mutación Missense , Miofibrillas/metabolismo , Subfragmentos de Miosina/metabolismo , Conejos , Tropomiosina/química , Troponina/metabolismoRESUMEN
Two-dimensional (2D) substrate rigidity promotes myosin II activity to increase traction force in a process negatively regulated by tropomyosin (Tpm) 2.1. We recently discovered that actomyosin contractility can increase intracellular pressure and switch tumor cells from low-pressure lamellipodia to high-pressure lobopodial protrusions during three-dimensional (3D) migration. However, it remains unclear whether these myosin II-generated cellular forces are produced simultaneously, and by the same molecular machinery. Here we identify Tpm 1.6 as a positive regulator of intracellular pressure and confirm that Tpm 2.1 is a negative regulator of traction force. We find that Tpm 1.6 and 2.1 can control intracellular pressure and traction independently, suggesting these myosin II-dependent forces are generated by distinct mechanisms. Further, these tropomyosin-regulated mechanisms can be integrated to control complex cell behaviors on 2D and in 3D environments.
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Miosina Tipo II/fisiología , Tropomiosina/fisiología , Citoesqueleto de Actina/fisiología , Actomiosina/fisiología , Movimiento Celular , Proteínas del Citoesqueleto , Matriz Extracelular , Fibroblastos/metabolismo , Prepucio/metabolismo , Humanos , Masculino , Miosina Tipo II/metabolismo , Presión , Cultivo Primario de Células , Seudópodos/fisiología , Tracción , Tropomiosina/metabolismoRESUMEN
INTRODUCTION: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. METHODS: Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. RESULTS: We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. CONCLUSIONS: We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.
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Neoplasias Encefálicas/fisiopatología , Glioblastoma/fisiopatología , Invasividad Neoplásica , Tropomiosina/fisiología , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Matriz Extracelular , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Hidrogeles , Ratones , Microscopía de Fuerza Atómica , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Esferoides Celulares/fisiología , Tropomiosina/genética , Tropomiosina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.
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Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Cardiomiopatía Dilatada/patología , Tropomiosina/fisiología , Animales , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/metabolismo , Células Cultivadas , Ratones , Ratones Transgénicos , Mutación , Miofibrillas/metabolismo , Miofibrillas/patología , FosforilaciónRESUMEN
Tropomyosin, one of the major actin filament-binding proteins, regulates actin-myosin interaction and actin-filament stability. Multicellular organisms express a number of tropomyosin isoforms, but understanding of isoform-specific tropomyosin functions is incomplete. The nematode Caenorhabditis elegans has a single tropomyosin gene, lev-11, which has been reported to express four isoforms by using two separate promoters and alternative splicing. Here, we report a fifth tropomyosin isoform, LEV-11O, which is produced by alternative splicing that includes a newly identified seventh exon, exon 7a. By visualizing specific splicing events in vivo, we find that exon 7a is predominantly selected in a subset of the body wall muscles in the head, while exon 7b, which is the alternative to exon 7a, is utilized in the rest of the body. Point mutations in exon 7a and exon 7b cause resistance to levamisole--induced muscle contraction specifically in the head and the main body, respectively. Overexpression of LEV-11O, but not LEV-11A, in the main body results in weak levamisole resistance. These results demonstrate that specific tropomyosin isoforms are expressed in the head and body regions of the muscles and contribute differentially to the regulation of muscle contractility.
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Contracción Muscular/fisiología , Tropomiosina/metabolismo , Tropomiosina/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Empalme Alternativo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Exones , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Empalme del ARN/genéticaRESUMEN
The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 ß-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.
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Citoesqueleto de Actina/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Tropomiosina/antagonistas & inhibidores , Citoesqueleto de Actina/efectos de los fármacos , Animales , Glucosa/administración & dosificación , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Tropomiosina/fisiologíaRESUMEN
Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The middle part of the Tpm molecule has some specific features, such as the presence of noncanonical residues as well as a substantial gap at the interhelical interface, which are believed to destabilize a coiled-coil and impart structural flexibility to this part of the molecule. To study how the gap affects structural and functional properties of α-striated Tpm (the Tpm1.1 isoform that is expressed in cardiac and skeletal muscles) we replaced large conserved apolar core residues located at both sides of the gap with smaller ones by mutations M127A/I130A and M141A/Q144A. We found that in contrast with the stabilizing substitutions D137L and G126R studied earlier, these substitutions have no appreciable influence on thermal unfolding and domain structure of the Tpm molecule. They also do not affect actin-binding properties of Tpm. However, they strongly increase sliding velocity of regulated actin filaments in an in vitro motility assay and cause an oversensitivity of the velocity to Ca2+ similar to the stabilizing substitutions D137L and G126R. Molecular dynamics shows that the substitutions studied here increase bending stiffness of the coiled-coil structure of Tpm, like that of G126R/D137L, probably due to closure of the interhelical gap in the area of the substitutions. Our results clearly indicate that the conserved middle part of Tpm is important for the fine tuning of the Ca2+ regulation of actin-myosin interaction in muscle.
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Sustitución de Aminoácidos , Tropomiosina/química , Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Calcio/farmacología , Humanos , Simulación de Dinámica Molecular , Movimiento (Física) , Mutación Missense , Mutación Puntual , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Temperatura , Tropomiosina/genética , Tropomiosina/fisiologíaRESUMEN
The actin cytoskeleton provides not only the underpinning for cell architecture but also mechanical force and the ability to drive movement of cells and their organelles. It is tempting to think of it simply as a set of stable structural elements, but nothing could be further from the truth. The cells of our bodies are continually remodelling their architecture by responding to a range of imposed biomechanical forces and intracellular functional demands. Studies of the dynamic and functional properties of the actin cytoskeleton have been dominated by a focus on actin and the view that actin filaments are essentially 'generic'. However, the 'other' component of most actin filaments in animals - tropomyosin - is coming into prominence. With this discovery is the realisation that far from being generic, actin filaments have their own functional individuality provided to them by their associated tropomyosin. This is changing the way we understand and study the actin cytoskeleton and has delivered a new therapeutic opportunity in what had come to be considered a 'no-go zone'.
Asunto(s)
Citoesqueleto de Actina , Evolución Biológica , Tropomiosina/fisiología , AnimalesRESUMEN
Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin-Tpm3.1 and actin-Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin-Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an "actin-Tpm code" that allows for spatial and temporal sorting of actomyosin function in the cell.
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Miosina Tipo V/metabolismo , Tropomiosina/metabolismo , Tropomiosina/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Transporte Biológico , Proteínas del Citoesqueleto/metabolismo , Humanos , Melanocitos/metabolismo , Melanosomas/metabolismo , Ratones , Cadenas Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Unión Proteica , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.
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Actinas/metabolismo , Células Epiteliales Alveolares/metabolismo , Extractos Vegetales/toxicidad , Tropomiosina/fisiología , Células Epiteliales Alveolares/efectos de los fármacos , Línea Celular Transformada , Expresión Génica , Humanos , Uniones Intercelulares/metabolismo , Estrés Oxidativo , Extractos Vegetales/química , Factores Protectores , Estabilidad Proteica , Proteolisis , Humo , Nicotiana/química , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.
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Citoesqueleto/fisiología , Membrana Eritrocítica/ultraestructura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiología , Animales , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrina/química , Espectrina/fisiología , Tropomiosina/química , Tropomiosina/fisiologíaRESUMEN
ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.
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Citoesqueleto de Actina/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Tropomiosina/fisiología , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.
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Miosina Tipo II/metabolismo , Tropomiosina/fisiología , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Movimiento Celular , Humanos , Subfragmentos de Miosina/fisiología , Ratas , Tropomiosina/químicaRESUMEN
Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ~5-fold more weakly than Tmod1 to α/ßTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/ßTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet ß/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate ß/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic ß- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-ß/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester ß- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.
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Actinas/fisiología , Isoformas de Proteínas/fisiología , Tropomodulina/fisiología , Tropomiosina/fisiología , Actinas/metabolismo , Animales , Citoesqueleto/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Unión Proteica , Isoformas de Proteínas/metabolismo , Conejos , Sarcómeros/metabolismo , Espectrometría de Fluorescencia , Tropomodulina/metabolismo , Tropomiosina/metabolismoRESUMEN
Tropomyosin is a two chained α-helical coiled coil protein that binds actin filaments and interacts with various actin binding proteins. Tropomyosin function depends on its ability to move to distinct locations on the surface of actin in response to the binding of different thin filament effectors. Tropomyosin dynamics plays an important role in these fluctuating interactions with actin and is thought to be fundamental to many of its biological activities. For example tropomyosin concerted movement on the surface of actin triggered by Ca(2+) binding to troponin or myosin head binding to actin has been argued to be key to the cooperative allosteric regulation of muscle contraction. These large-scale motions are affected by tropomyosin internal dynamics and mechanical properties. Tropomyosin internal dynamics corresponding to smaller and more localised structural fluctuations are increasingly recognised to play an important role in its function. A thorough understanding of the coupling between local and global structural fluctuations in tropomyosin is required to understand how time dependent structural fluctuations in tropomyosin contribute to the overall thin filament dynamics and dictate their various biological activities.
Asunto(s)
Movimiento/fisiología , Músculo Esquelético/fisiología , Tropomiosina/fisiología , Citoesqueleto de Actina/fisiología , Animales , Humanos , Contracción Muscular/fisiologíaRESUMEN
Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.