RESUMEN
The origins of the large differences observed in the rates at which diverse particles are conveyed along axonal microtubules are still a matter of debate in the literature. There is evidence that certain neurodegenerative diseases may be triggered by disturbances in the related transport processes. Motivated by this, we employ a model to investigate mobility properties of certain cargoes whose dynamics are coupled with that of molecular motors on crowded microtubules. For certain initial and boundary conditions, we use the method of characteristics to resolve perturbatively the pair of equations of Burgers type resulting from a mean-field approach to the original microscopic stochastic model. Extensions to nonperturbative limits are explored numerically. In this context, we are able to figure out conditions under which the cargoes' average velocities may differ up to orders of magnitude just by changing the number of motors on the considered track. We then discuss possibilities to connect these theoretical predictions with available experimental data about axon transport.
Asunto(s)
Transporte Axonal , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Microtúbulos/metabolismoRESUMEN
Molecular motors are fascinating proteins that use the energy of ATP hydrolysis to drive vesicles and organelles along cytoskeleton filaments toward their final destination within the cell. Several copies of these proteins bind to the cargo and take turns transporting the cargo attaching to and detaching from the track stochastically. Despite the relevance of molecular motors to cell physiology, key aspects of their collective functioning are still unknown. In this work we propose a one-dimensional model for the transport of extensive and smooth organelles driven by molecular motors. We ran numerical simulations to study the behavior of the cargo for different motor configurations, focusing on the transport properties observable in the experiments, e.g., average speed of the organelle and variations in length. We found that active motors drive the cargo using two different mechanisms: Either they locate in front of the cargo and pull the organelle or they situate at the cargo lagging edge and push. Variations in the organelle length is in close relation with the fraction of motors in each configuration, which depends on the resisting load. The results of this model were contrasted with experimental data obtained from the tracking of rodlike mitochondria during active transport in Xenopus laevis melanophores.
Asunto(s)
Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Orgánulos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Biomecánicos , Melanóforos/metabolismo , Xenopus laevisRESUMEN
Following entry into the host cell, retroviruses generate a dsDNA copy of their genomes via reverse transcription, and this viral DNA is subsequently integrated into the chromosomal DNA of the host cell. Before integration can occur, however, retroviral DNA must be transported to the nucleus as part of a 'preintegration complex' (PIC). Transporting the PIC through the crowded environment of the cytoplasm is challenging, and retroviruses have evolved different mechanisms to accomplish this feat. Within a eukaryotic cell, microtubules act as the roads, while the microtubule-associated proteins dynein and kinesin are the vehicles that viruses exploit to achieve retrograde and anterograde trafficking. This review will examine the various mechanisms retroviruses have evolved in order to achieve retrograde trafficking, confirming that each retrovirus has its own strategy to functionally subvert microtubule associated proteins.
Asunto(s)
Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Retroviridae/fisiología , Transporte Biológico , Dineínas/química , Dineínas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Replicación ViralRESUMEN
Lysosomes are dynamic organelles, which can fuse with a variety of targets and undergo constant regeneration. They can move along microtubules in a retrograde and anterograde fashion by using motor proteins, kinesin and dynein, being main players in extracellular secretion, intracellular components degradation and recycling. Moreover, lysosomes interact with other intracellular organelles to regulate their turnover, such as ER, mitochondria and peroxisomes. The correct localization of lysosomes is relevant in several physiological processes, including appropriate antigen presentation, neurotransmission and receptors modulation in neuronal synapsis, whereas hepatic lysosomes and autophagy are master regulators of nutrient homeostasis. Alterations in lysosome function due to mutation of genes encoding lysosomal proteins, soluble hydrolases as well as membrane proteins, lead to lysosomal storage diseases (LSDs). Lysosomes containing undegraded substrates are finally stacked and therefore miss positioned inside the cell, leading to lysosomal dysfunction, which impacts a wide range of cellular functions.
Asunto(s)
Movimiento Celular , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Redes y Vías Metabólicas/genética , Modelos Biológicos , Mutación , Proteínas/genéticaRESUMEN
One-dimensional models coupling a Langevin equation for the cargo position to stochastic stepping dynamics for the motors constitute a relevant framework for analyzing multiple-motor microtubule transport. In this work we explore the consistence of these models focusing on the effects of the thermal noise. We study how to define consistent stepping and detachment rates for the motors as functions of the local forces acting on them in such a way that the cargo velocity and run-time match previously specified functions of the external load, which are set on the base of experimental results. We show that due to the influence of the thermal fluctuations this is not a trivial problem, even for the single-motor case. As a solution, we propose a motor stepping dynamics which considers memory on the motor force. This model leads to better results for single-motor transport than the approaches previously considered in the literature. Moreover, it gives a much better prediction for the stall force of the two-motor case, highly compatible with the experimental findings. We also analyze the fast fluctuations of the cargo position and the influence of the viscosity, comparing the proposed model to the standard one, and we show how the differences on the single-motor dynamics propagate to the multiple motor situations. Finally, we find that the one-dimensional character of the models impede an appropriate description of the fast fluctuations of the cargo position at small loads. We show how this problem can be solved by considering two-dimensional models.
Asunto(s)
Transporte Biológico/fisiología , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Temperatura , Simulación por Computador , Método de Montecarlo , Movimiento (Física) , Procesos EstocásticosRESUMEN
BACKGROUND AND OBJECTIVES: In this study, the aim was to compare postoperative analgesia effects of the administration of ultrasound-guided interscalene brachial plexus block and intra-articular bupivacaine carried out with bupivacaine. METHODS: In the first group of patients 20 mL 0.25% bupivacaine and ultrasound-guided interscalene brachial plexus block (ISPB) were applied, while 20 mL 0.25% bupivacaine was given via intra-articular (IA) administration to the second group patients after surgery. Patients in the third group were considered the control group and no block was performed. Patient-controlled analgesia (PCA) with morphine was used in all three groups for postoperative analgesia. RESULTS: In the ISPB group, morphine consumption in the periods between 0-4, 6-12 and 12-24 postoperative hours and total consumption within 24 h was lower than in the other two groups. Morphine consumption in the IA group was lower than in the control group in the period from 0 to 6 h and the same was true for total morphine consumption in 24 h. Postoperative VASr scores in the ISPB group were lower than both of the other groups in the first 2 h and lower than the control group in the 4th and 6th hours (p < 0.05). In the IA group, VASr and VASm scores in the 2nd, 4th and 6th hours were lower than in the control group (p < 0.05). CONCLUSION: Interscalene brachial plexus block was found to be more effective than intra-articular local anesthetic injection for postoperative analgesia. .
JUSTIFICATIVA E OBJETIVOS: Comparar os efeitos na analgesia no pós-operatório da administração de bloqueio do plexo braquial por via interescalênica guiado por ultrassom e bupivacaína intra-articular, feito com bupivacaína. MÉTODOS: No primeiro grupo de pacientes, 20 mL de bupivacaína a 0,25% e bloqueio do plexo braquial por via interescalênica guiado por ultrassom (BPBI) foram administrados, enquanto 20 mL de bupivacaína a 0,25% foram administrados por via intra-articular (IA) ao segundo grupo de pacientes após a cirurgia. Os pacientes do terceiro grupo foram considerados grupo controle e nenhum bloqueio foi feito. Analgesia controlada pelo paciente (ACP) com morfina foi usada nos três grupos para analgesia pós-operatória. RESULTADOS: No grupo BPBI, o consumo de morfina nos períodos entre 0-4, 6-12 e 12-24 horas após a cirurgia e o consumo total em 24 horas foram mais baixos do que nos outros dois grupos. O consumo de morfina no grupo IA foi menor do que no grupo controle no período de 0-6 horas, como também foi menor o consumo total de morfina em 24 horas. Os escores EVAr no pós-operatório do grupo BPBI foram menores do que os escores dos dois outros grupos nas primeiras duas horas e menores do que os do grupo controle nos períodos de 4 e 6 horas (p < 0,05). No grupo IA, os escores EVAr e EVAm nos períodos de 2, 4 e 6 horas foram menores do que no grupo controle (p < 0,05). CONCLUSÃO: O bloqueio do plexo braquial por via interescalênica mostrou ser mais eficaz do que a injeção intra-articular de anestésico local para analgesia pós-operatória. .
JUSTIFICACIÓN Y OBJETIVOS: En este estudio, nuestro objetivo fue comparar en el período postoperatorio los efectos analgésicos de la administración de la bupivacaína en el bloqueo del plexo braquial por vía interescalénica guiado por ecografía y bupivacaína intraarticular. MÉTODOS: En el primer grupo de pacientes se administraron 20 mL de bupivacaína al 0,25% y se llevó a cabo el bloqueo del plexo braquial por vía interescalénica (BPBI) guiado por ecografía, mientras que al segundo grupo de pacientes se le administraron 20 mL de bupivacaína al 0,25% por vía intraarticular (IA) tras la cirugía. Los pacientes del tercer grupo fueron considerados como grupo control y en ellos no se realizó ningún bloqueo. La analgesia controlada por el paciente con morfina se usó en los 3 grupos para la analgesia postoperatoria. RESULTADOS: En el grupo BPBI, el consumo de morfina en los períodos entre 0-4, 6-12 y 12-24 h del postoperatorio y el consumo total en 24 h fueron más bajos que en los otros 2 grupos. El consumo de morfina en el grupo IA fue menor que en el grupo control en el período de 0-6 h, como también fue menor el consumo total de morfina en 24 h. Las puntuaciones EVAr en el postoperatorio del grupo BPBI fueron menores que las de los otros 2 grupos en las primeras 2 h y menores que los del grupo control en los períodos de 4 y 6 h (p < 0,05). En el grupo IA, las puntuaciones EVAr y EVAm en los períodos de 2, 4 y 6 h fueron menores que en el grupo control (p < 0,05). CONCLUSIÓN: El BPBI mostró ser más eficaz que la inyección intraarticular de anestésico local para analgesia postoperatoria. .
Asunto(s)
Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dineínas/química , Dineínas/aislamiento & purificación , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
We study theoretically and by numerical simulations the motion of particles driven by molecular motors in a viscoelastic medium representing the cell cytoplasm. For this, we consider a generalized Langevin equation coupled to a stochastic stepping dynamics for the motors that takes into account the action of each motor separately. In the absence of motors, the model produces subdiffusive motion of particles characterized by a power-law scaling of the mean square displacement versus the lag time as t^{α}, with 0<α<1, similar to that observed in cells. Our results show how the action of the motors can induce a transition to a superdiffusive regime at large lag times with the characteristics of those found in experiments reported in the literature. We also show that at small lag times, the motors can act as static crosslinkers that slow down the natural subdiffusive transport. An analysis of previously reported experimental data in the relevant time scales provides evidence of this phenomenon. Finally, we study the effect of a harmonic potential representing an optical trap, and we show a way to approach to a macroscopic description of the active transport in cells. This last point stresses the relevance of the molecular motors for generating not only directed motion to specific targets, but also fast diffusivelike random motion.
Asunto(s)
Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Movimiento (Física) , Sustancias Viscoelásticas/metabolismo , Simulación por Computador , Citoplasma/metabolismo , Difusión , Orgánulos/metabolismo , Procesos EstocásticosRESUMEN
BACKGROUND: Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment. METHODS: We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors. RESULTS AND CONCLUSIONS: Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion. GENERAL SIGNIFICANCE: Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Tamaño de los Orgánulos/fisiología , Orgánulos/fisiología , Actinas/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Microambiente Celular/fisiología , Difusión , Dineínas/metabolismo , Filamentos Intermedios/metabolismo , Melanóforos/metabolismo , Melanóforos/fisiología , Melanosomas/metabolismo , Melanosomas/fisiología , Microtúbulos/metabolismo , Microtúbulos/fisiología , Orgánulos/metabolismo , Relación Estructura-Actividad , Xenopus laevisRESUMEN
The presence of protein aggregates is common in neurodegenerative disorders; however, the real cause and effect of these aggregates during neurodegeneration is still a matter of investigation. We hypothesize that impairment of intracellular traffic may appear in the absence of protein inclusions and might trigger protein aggregation. In the present study, we aimed to evaluate mitochondria mobility as well as protein and messenger RNA expression of KIF1B and KIF5 that are molecular motors for neuronal anterograde traffic, in hippocampus, substantia nigra, and locus coeruleus of 10-month-old Lewis rats and cultured cells, from these same areas, following exposure to low doses of rotenone that do not lead to protein inclusions. The present study showed alteration in KIF1B and KIF5 expression, as well as in mitochondria mobility prior to protein aggregation involved in neurodegenerative disorders. These findings suggest that change in intracellular trafficking might be critical and one of the primary events for impairment of cell physiology during neurodegeneration associated with protein inclusions.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Mitocondrias/metabolismo , Proteínas Motoras Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/mortalidad , Rotenona/farmacología , Envejecimiento/efectos de los fármacos , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Cinesinas/genética , Cinesinas/metabolismo , Mitocondrias/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
The adaptation of GH(3) cells to different microenvironments is a consequence of a partial compromise with the tumor phenotype. A collagen type IV enriched microenvironment favors an invasive phenotype and increases the substrate adhesion capacity, whereas it decreases the phosphorylation of the regulatory myosin light chain and the aggregation capacity. In contrast, the higher internal tension and increased aggregation capacity induced by collagen type I/III are factors that reduce the invasion rate. Our results show, for the first time, the importance of collagen subtypes in determining the migratory strategy: collagen I/III favors mesenchymal-like motility, whereas collagen type IV induces an ameboid-type displacement. The reciprocal modulation of the myosin light chain kinase and the Rho-kinase determines the invasive capacity through changes in tissue cohesion, extracellular matrix affinity, regulatory myosin light chain phosphorylation and spatial distribution. The collagen subtype determines which of the mechano-transduction signaling pathways will regulate the tensional homeostasis and affect the invasion ability as well as the preferred migration strategy of the cells.
Asunto(s)
Adhesión Celular/fisiología , Colágeno/metabolismo , Invasividad Neoplásica/fisiopatología , Microambiente Tumoral/fisiología , Actomiosina/metabolismo , Adenoma/patología , Adenoma/fisiopatología , Animales , Agregación Celular/fisiología , Línea Celular Tumoral , Colágeno/clasificación , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Invasividad Neoplásica/patología , Neoplasias Hipofisarias/patología , Neoplasias Hipofisarias/fisiopatología , Ratas , Transducción de Señal , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Here we describe a novel component essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded by motF (RSP_0067), the first gene of a predicted transcriptional unit which contains two hypothetical genes. Sequence analysis indicated that MotF is a bitopic membrane-spanning protein. Protease sensitivity assays and green fluorescent protein (GFP) fusions confirmed this prediction and allowed us to conclude that the C terminus of MotF is located in the periplasmic space. Wild-type cells expressing a functional GFP-MotF fusion show a single fluorescent focus per cell. The localization of this protein in different genetic backgrounds allowed us to determine that normal localization of MotF depends on the presence of FliL and MotB. Characterization of a ΔmotF pseudorevertant strain revealed that a single nucleotide change in motB suppresses the Mot(-) phenotype of the motF mutant. Additionally, we show that MotF also becomes dispensable when other mutant alleles of motB previously isolated as second-site suppressors of ΔfliL were expressed in the motF mutant strain. These results show that MotF is a new component of the Fla1 flagellum, which together with FliL is required to promote flagellar rotation, possibly through MotB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Motoras Moleculares/metabolismo , Rhodobacter sphaeroides/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos , Proteínas Bacterianas/genética , Femenino , Flagelos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Motoras Moleculares/genética , Mutación , Periplasma , Plásmidos , Mutación Puntual , Rhodobacter sphaeroides/genéticaRESUMEN
In this work, we explored theoretically the transport of organelles driven along microtubules by molecular motors of opposed polarities using a stochastic model that considers a Langevin dynamics for the cargo, independent cargo-motor linkers and stepping motion for the motors. It has been recently proposed that the stiffness of the motor plays an important role when multiple motors collectively transport a cargo. Therefore, we considered in our model the recently reported values for the stiffness of the cargo-motor linker determined in living cells (â¼0.01 pN/nm,) which is significantly lower than the motor stiffness obtained in in vitro assays and used in previous studies. Our model could reproduce the multimodal velocity distributions and typical trajectory characteristics including the properties of the reversions in the overall direction of motion observed during melanosome transport along microtubules in Xenopus laevis melanophores. Moreover, we explored the contribution of the different motility states of the cargo-motor system to the different modes of the velocity distributions and could identify the microscopic mechanisms of transport leading to trajectories compatible with those observed in living cells. Finally, by changing the attachment and detachment rates, the model could reproduce the different velocity distributions observed during melanosome transport along microtubules in Xenopus laevis melanophores stimulated for aggregation and dispersion. Our analysis suggests that active tug-of-war processes with loose mechanical coupling can account for several aspects of cargo transport along microtubules in living cells.
Asunto(s)
Fenómenos Mecánicos , Melanosomas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Animales , Transporte Biológico , Fenómenos Biomecánicos , Cinética , Proteínas Motoras Moleculares/metabolismo , Movimiento , Procesos Estocásticos , Xenopus laevisRESUMEN
We propose a biochemical model providing the kinetic and energetic descriptions of the processivity dynamics of kinesin and dinein molecular motors. Our approach is a modified version of a well known model describing kinesin dynamics and considers the presence of a competitive inhibition reaction by ADP. We first reconstruct a continuous free-energy landscape of the cycle catalyst process that allows us to calculate the number of steps given by a single molecular motor. Then, we calculate an analytical expression associated to the translational velocity and the stopping time of the molecular motor in terms of time and ATP concentration. An energetic interpretation of motor processivity is discussed in quantitative form by using experimental data. We also predict a time duration of collective processes that agrees with experimental reports.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Biocatálisis , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Cinética , Modelos Moleculares , Proteínas Motoras Moleculares/antagonistas & inhibidores , Biosíntesis de Proteínas , TermodinámicaRESUMEN
We analyze theoretically the problem of cargo transport along microtubules by motors of two species with opposite polarities. We consider two different one-dimensional models previously developed in the literature: a quite widespread model which assumes equal force sharing, here referred to as the mean field model (MFM), and a stochastic model (SM) which considers individual motor-cargo links. We find that in generic situations, the MFM predicts larger cargo mean velocity, smaller mean run time and less frequent reversions than the SM. These phenomena are found to be the consequences of the load sharing assumptions and can be interpreted in terms of the probabilities of the different motility states. We also explore the influence of the viscosity in both models and the role of the stiffness of the motor-cargo links within the SM. Our results show that the mean cargo velocity is independent of the stiffness, while the mean run time decreases with such a parameter. We explore the case of symmetric forward and backward motors considering kinesin-1 parameters, and the problem of transport by kinesin-1 and cytoplasmic dyneins considering two different sets of parameters previously proposed for dyneins.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Transporte Biológico , Microtúbulos/metabolismo , Modelos Biológicos , Procesos EstocásticosRESUMEN
The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is â¼ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.
Asunto(s)
Fenómenos Mecánicos , Melanosomas/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Fenómenos Biomecánicos , Supervivencia Celular , Complejo Dinactina , Elasticidad , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Transporte de Proteínas , Viscosidad , Xenopus laevisRESUMEN
In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Motoras Moleculares/metabolismo , Receptores de Mineralocorticoides/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Dineínas/química , Dineínas/metabolismo , Humanos , Inmunofilinas/química , Inmunofilinas/metabolismo , Ratones , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/química , Complejos Multiproteicos , Células 3T3 NIH , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Ratas , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Unión a Tacrolimus/deficiencia , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2-8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis.
Asunto(s)
Meiosis , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Oocitos/enzimología , Oogénesis , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Ensamble y Desensamble de Cromatina , Citoplasma/metabolismo , Complejo Dinactina , Dineínas/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Meiosis/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Proteínas Motoras Moleculares/antagonistas & inhibidores , Nocodazol/farmacología , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Transporte de Proteínas , Factores de Tiempo , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Vanadatos/farmacologíaRESUMEN
It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fcgamma-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism for membrane recycling. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the cytoskeletal requirements are largely unknown. To study this vesicle-mediated recycling transport, we used a biochemical assay and we developed a method to follow this process by confocal fluorescence microscopy. Interestingly, recycling from the phagosomal compartment was increased when the actin cortex was thinned by inhibitors of F-actin. In contrast, depolymerization of microtubules diminished both phagocytosis and recycling from phagosomes. Our results suggest that actin and microtubules are needed not only for phagosome biogenesis but also at other steps along the phagocytic pathway.