CONICET | Buscador de Institutos y Recursos Humanos

The organization of the cytoplasm is regulated by molecular motors that transport organelles and other cargoes along microtubules and actin filaments. The cellular environment is also viscoelastic causing viscous drag and elastic tethering to surrounding structures. Although it is known that cargoes are propelled in cells by groups of cytoskeleton motors, the precise impact of collective motors behaviors on intracellular transport and trafficking remains controversial. Novel single particle tracking techniques allow recovering the trajectories of single organelles while they move within the cytoplasm with nanometer and millisecond resolution. These trajectories typically consist of series of back and forth movements called ?runs?, interspersed with periods of stationary or diffusive motion. Since organelles are driven by multiple copies of motors, the statistical analysis of their trajectories can reveal key aspects of the collective behavior of motors in living cell, as well as of the complex relation between organelle motion and microenvironment [1-4]. Recently, we used a fast and precise single particle tracking method to follow the motion of individual melanosomes (pigmented organelles) in the cell cytoplasm of Xenopus laevis melanophores. We found that small organelles, which are supposed to experience a smaller drag force, present more tortuous trajectories and found that the actin and intermediate filament networks play important roles in this behavior. On the other hand, the transport of big organelles was less influenced by the cytoskeletal environment and trajectories were more processive, suggesting that the number of copies of active motors increases with organelle size. To further explore the mechanisms that regulate the activity of motor proteins, we tracked fluorescently tagged peroxisomes, which are organelles involved in the catabolism of fatty acids, in Drosophila melanogaster S2 cells. These cells can be induced to form long processes filled with uniformly orientated microtubules, making them a privilege system for the study of organelle transport since the motion is quasi 1- dimensional. Peroxisomes are driven along microtubules within the processes by the action of the motor proteins kinesin-1 (motion toward the processes tip) and cytoplasmic dynein (motion toward the perinuclear region). The analysis of the run- lengths and segmental velocities of peroxisomes suggested that dynein motors would regulate anterograde transport driven by kinesin motors. To explore how the biophysical properties of motors affect the transport, we extended the analysis to peroxisomes trajectories in cells expressing a mutant motor Eg5, which has similar stall force and step size than those of kinesin-1, but is slower. In this talk I will first give a short introduction to intracellular transport mediated by molecular motors. Then, I will describe the experiments that we performed in our lab to study the different aspects of the transport. I will focus on our recent experiments on melanophores and S2 cells and the interpretation of our results in terms of a model of interacting motors. References: 1. Exchange of microtubule molecular motors during melanosome transport in Xenopus laevis melanophores is triggered by collisions with intracellular obstacles. Luciana Bruno, María M. Echarte, and Valeria Levi. Cell Biochemistry and Biophysics. Vol 52, Number 3: 191-201 (2008). 2. Mechanical properties of organelles driven by microtubule-dependent molecular motors in living cells. Luciana Bruno, Marcelo Salierno, Diana Wetzler, Marcelo A. Desposito, Valeria Levi. PLoS ONE 6(4): e18332. (2011) 3. Transport Properties of Melanosomes along Microtubules Interpreted by a Tug-of-War Model with Loose Mechanical Coupling. Sebastián Bouzat, Valeria Levi, Luciana Bruno. PLoS ONE 7(8): e43599. (2012). 4 . When size does matter: organelle size influences the properties of transport mediated by molecular motors. María C De Rossi; Luciana Bruno, Alejandro Wolosiuk, Marcelo A Despósito, Valeria Levi. Biochimica et Biophysica Acta 1830 5095?5103 (2013).