title: Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport.
abstract: Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.
authors.affiliation: Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, Texas.
authors.forename: Christina
authors.initials: C
authors.lastname: Leidel
authors.affiliation: Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, Texas.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Longoria
authors.affiliation: Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, Texas.
authors.forename: Franciso Marquez
authors.initials: FM
authors.lastname: Gutierrez
authors.affiliation: Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, Texas; Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas. Electronic address: shubeita@chaos.utexas.edu.
authors.forename: George T
authors.initials: GT
authors.lastname: Shubeita
mesh_terms: D000818:Animals; D001692:Biological Transport; D004331:Drosophila melanogaster / Q000196:embryology; D004625:Embryo, Nonmammalian / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000378:metabolism*; D052898:Optical Tweezers; D013997:Time Factors
chemical_list: D020409:Molecular Motor Proteins