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Activité motrice de myosines dans des réseaux de filaments d’actine d’architecture contrôlée in vitro

Abstract : Molecular motors navigate the cytoskeleton to position vesicles and organelles at specific locations in the cell. Cytoskeletal filaments assemble into parallel, antiparallel or disordered networks, providing a complex environment that constrains active transport properties. Using surface micro-patterns of nucleation-promoting factors to control the geometry of actin polymerization, we studied in vitro the interplay between the actin-network architec-ture and cargo transport by small myosin assemblies. With two parallel nucleation lines, we produced an antiparallel network of overlapping filaments. We found that 200nm beads coated with processive myosin V motors displayed directed movements towards the mid-line of the pattern, where the net polarity of the actin network was null, and accumulated there. The bead distribution was dictated by the spatial profiles of bead velocity and diffu-sion coefficient, indicating that a diffusion-drift process was at work. Interestingly, beads coated with skeletal heavy mero-myosin II motors showed a similar behavior. However, although velocity gradients were sharper with myosin II, the much larger bead diffusion observed with this non-processive motor resulted in less precise positioning. Strikingly, bead positioning did not depend on the spacing between the nucleation lines. Our observa-tions are well described by a three-state model of bead transport, in which active beads locally sense the net polarity of the filament network by frequently detaching from and re-attaching to the filaments. A stochastic sequence of processive runs and diffusive search-es results in a biased random walk with an effective drift velocity and diffusion coefficient. Positioning relies on spatial gradients of the net actin polarity, as well as on the run length of the cargo in the attached state. Altogether, our results on a minimal acto-myosin system demonstrate the key role played by the actin-network architecture on motor transport. Molecular motors can also deform and reorganize the cytoskeleton. Adding heavy mero-myosin II or V in bulk, we observed that parallel networks of actin filaments emerg-ing from a nucleation line or disk can self-assemble into bundles that beat periodically like the flagellum of the spermatozoid. In a preliminary analysis, we observed waves of defor-mation travelling from the base to the tip of the bundle at a speed of 0,5 µm/s. As time went by, the bundles grew thicker, resulting in an increase of the beating period (range: 25-40 s). In addition, neighboring actin ‘flagella’ were able to synchronize, as observed in vivo for instance with the the two flagella of the algae Chlamydomonas. Our minimal acto-myosin system thus mimicked key properties of microtubule-based flagellar beating, de-spite the different nature of the motors and cytoskeletal filaments involved. This system thus provides a new tool to study the generic physical properties of flagellar beating.
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Submitted on : Thursday, March 15, 2018 - 12:15:09 PM
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Mathieu Richard. Activité motrice de myosines dans des réseaux de filaments d’actine d’architecture contrôlée in vitro. Biophysique []. Université Sorbonne Paris Cité, 2016. Français. ⟨NNT : 2016USPCB054⟩. ⟨tel-01734989⟩



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