Descripción: 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 ~ 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. © 2011 Bruno et al.

Descripción: 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 (~0.01 pN/nm, [1]) 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. © 2012 Bouzat et al.

Descripción: Electrical transmission among neurons has been considered a mechanism to synchronize neuronal activity, and rectification provides a mechanism to confine the flow of signals among the connected neurons. The question is how this type of transmission operates within complex neuronal networks. In the leech, the neurons located in position 151 of the midbody ganglion map are connected to virtually every motoneuron via rectifying electrical synapses that pass negative current to the motoneurons. These are nonspiking neurons, and here we have labeled them NS neurons. The goal of this investigation has been to assess their role in regulating motor activity and how rectifying electrical synapses contribute to the function of motor networks. The coupling between NS neurons and motoneurons was voltage sensitive: it increased as motoneurons were depolarized. In addition, excitation of motoneurons evoked hyperpolarizing synaptic responses in NS neurons, the amplitude of which depended on the membrane potential of the latter and on the motoneuron firing frequency. This hyperpolarization was mediated by chemical transmission through an interneuronal layer that spanned the nerve cord. These interactions established a feedback loop between NS and motoneurons that was regulated by the membrane potential of NS. This mechanism was responsible for the uncoupling between otherwise electrically coupled motoneurons. In this way, the NS neurons can act as "electrical neuromodulators," modifying the interaction of other neurons, depending on the activity of the system as a whole.

Descripción: Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR-hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR·hsp90·immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR - L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.

Descripción: 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. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Descripción: Although cyclophilin A (CyP-A) is a relatively abundant small immunophilin present in the cytoplasm of all mammalian cells, its general function(s) in the absence of the immunosuppressant drug cyclosporin A is not known. In contrast, the high molecular weight hsp90-binding immunophilins appear to play a role in protein trafficking in that they have been shown to link glucocorticoid receptor-hsp90 and p53-hsp90 complexes to the dynein motor protein for retrograde movement along microtubules. These immunophilins link to cytoplasmic dynein indirectly through the association of the immunophilin peptidylprolyl isomerase (PPIase) domain with dynamitin, a component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Here, we show that CyP-A exists in native heterocomplexes containing cytoplasmic dynein that can be formed in cell-free systems. Prolyl isomerase activity is not required for forming the dynein complex, but the PPIase domain fragment of FKBP52 blocks complex formation and CyP-A binds to dynamitin in a PPIase domain-dependent manner. CyP-A heterocomplexes containing tubulin and dynein can be formed in cytosol prepared under microtubule-stabilizing conditions, and CyP-A colocalizes in mouse fibroblasts with microtubules. Colocalization with microtubules is disrupted by overexpression of the PPIase domain fragment. Thus, we conclude that CyP-A associates in vitro and in vivo with the dynein/dynactin motor protein complex and we suggest that CyP-A may perform a general function related to the binding of cargo for retrograde movement along microtubules.

Descripción: The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend -70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V. © 2009 by the Biophysical Society.