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Modeling Cargo Transport by Pairs of Kinesin-1 and -3 Motors
Monday, February 13, 2017 contribute to force generation by single kinesin-1 motors under purified conditions and has been predicted to function in force generation across the kinesin superfamily. However, differences in coverstrand and necklinker sequences make the role of the CNB across different kinesin families unclear. We adapted artificial cargo systems to delineate key features of the CNB during multi-motor transport against low and high loads in cells. Our results indicate that in teams, kinesin-1 motors can robustly transport cargo against high- and low-load in cells. Molecular modeling was used to predict mutations in the coverstrand and necklinker that disrupt and shorten beta-sheet formation. Experimental testing of these mutations shows that impaired beta-sheet formation compromised the capacity for teams of kinesin-1 motors to transport high-load cargo but not low-load cargo to the periphery of cells. Surprisingly, truncation of the region N-terminal to the coverstrand, predicted to have a minimal role in CNB formation, severely crippled the capacity for teams of kinesin-1 motors to transport both low- and high-load cargoes in cells. We plan to extend these studies to additional kinesin families to identify distinct motility dependencies on formation of the CNB. 1289-Pos Board B357 Modeling Cargo Transport by Pairs of Kinesin-1 and -3 Motors Goker Arpag1, Stephen Norris2, Kristen Verhey3, William O. Hancock4, Erkan Tuzel5. 1 Physics, Worcester Polytechnic Institute, Worcester, MA, USA, 2Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA, 3Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA, 4 Bioengineering, Pennsylvania State University, University Park, PA, USA, 5 Physics, Worcester Polytechnic Institute, Worcester, MA, USA. Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we analyzed the transport properties of complexes of kinesin-1 and kinesin-3 motors attached to protein scaffolds in vitro (Norris et al. (2014) JCB 207:393-406). To uncover the underlying motor dynamics, a model of cargo transport was developed with motors connected to the cargo through elastic tethers and motor stepping rates and force-dependent detachment rates taken from previous work (Arpag et al. (2014) Biophys. J. 107:1896-1904). Predicted mult-motor velocities were found to be strongly dependent on the rate that motors reattach to the microtubule following detachment. From single-motor landing rate experiments, kinesin-3 was found to have a 3-fold higher landing rate than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this ‘‘K-loop’’ is a key determinant of motor (re)attachment rate. Simulations of these loopswapped motors, carried out using the experimentally determined relative on-rates, matched the experimental results. Thus, the transport behavior of cargo carried by pairs of kinesin-1 -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load-dependence of detachment, and the motor on-rate. This quantitative characterization of cargo transport by pairs of dissimilar motors provides a step toward understanding multi-motor transport in cells. 1290-Pos Board B358 Engineering Inhibitable Kinesin-3 Motors by a Novel Chemical-Genetic Approach Shirley Chen, T. Lynne Blasius, Martin F. Engelke, Kristen J. Verhey. Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. Kinesins are a superfamily of microtubule-dependent motor proteins that play a fundamental role in the development and maintenance of cell size, shape, and organization. 45 genes in the human genome encode for kinesin proteins belonging to 16 different families, with the adult brain expressing at least half of these genes. Kinesin transport is particularly important in neurons because of this cell type’s highly polarized structure and the long distances that cargoes are required to travel. In fact, many neurodegenerative diseases demonstrate defects in axonal transport. One kinesin motor implicated in neuronal transport is KIF1A, a member of the kinesin-3 family. KIF1A is responsible for the transport of synaptic vesicle precursors from the cell body to the axon terminal. A major challenge to understanding kinesin function in cells, including neurons, is the absence of small molecule inhibitors specific to one kinesin motor. This probably results from the high sequence and structural conservation within the kinesin motor domain. Although cur-
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rent genetic techniques do allow disturbance of a specific kinesin, they do not act fast enough to distinguish between the direct and indirect consequences of a specific motor’s malfunction. Here we apply a novel chemical-genetic approach to engineer inhibitable KIF1A motors that function normally in the absence of a small, cell-permeable molecule but are then specifically and rapidly inhibited in the presence of the small molecule. Through this work, we have designed six inhibitable KIF1A motors, which we will further characterize through live cell and single molecule imaging. We also aim to use these motors to study the specific roles of kinesin-3 motors in axonal transport and neuronal disease. Furthermore, because of the kinesin motor domain’s high conservation, these results will be informative when designing inhibitable versions of other members of the kinesin superfamily.
Myosins 1291-Pos Board B359 Molecular Mechanism of Synchronous Force Generations among Myosin Molecules Motoshi Kaya1, Takumi Washio2, Hideo Higuchi1. 1 Dept. Physics, University of Tokyo, Tokyo, Japan, 2Frontier Science, University of Tokyo, Tokyo, Japan. For more than half a century, molecular mechanism of muscle contraction has been investigated by various experimental approaches. One of main questions is whether myosin molecules generate force cooperatively? To address the question, we have developed the experimental system, in which synthetic myofilaments interact with a single actin filament. Our findings suggest that myosin molecules may generate force synchronously against high loads. To gain insight into the mechanism of cooperative force generations, we developed the simulation model consisting of 17 myosin molecules arranged in series and interacting with a single actin filament. The model revealed that strain-dependent kinetics and multiple steps of power strike enhance a chance of synchronous force generations among myosin molecules. 1292-Pos Board B360 O-Myo! An O-Shaped Myosin Gliding Assay for Characterizing LongTerm Actin-Myosin Behaviors Rizal F. Hariadi1, Abhinav J. Appukutty2, Sivaraj Sivaramakrishnan3. 1 Department of Physics and the Biodesign Institute, Arizona State University, Tempe, AZ, USA, 2Department Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 3Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA. Nature has evolved molecular machines that are critical in cellular processes occurring over broad timescales, ranging from seconds to days. As an example, myosin in the human heart take ~10 steps per second and are collectively responsible for ~10 heart beats per day. Analogous to the engineering of macro-scale machines, the evolution of molecular machines is likely to be constrained by the requirements of high short-term performance and a long lifetime. Here, we developed a circular gliding assay (O-Myo) that utilizes engineered micron-scale DNA nanotube rings with defined geometrical arrangements of dimeric myosin VI, a model system for myosin motor. The O-Myo gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 myosin motors per ring) multiple times, once per ring revolution, providing a method of assessing the long-term dynamics of actin-myosin interactions. Actin filaments glide along the nanotube rings with high processivity for up to 5 full revolutions (40 mm total run length; 11 minutes), consistent with cooperative interactions between a single actin filament and multiple myosin motors. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 mm). As a model application of OMEGA, we then analyze motor-based mechanical influence on ‘‘stop-and-go’’ gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes. 1293-Pos Board B361 Changes in Myosin Crossbridge Cycle during Human Development Alice Ward Racca, Samantha Lynn, Michael A. Geeves. University of Kent, Canterbury, United Kingdom.
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rent genetic techniques do allow disturbance of a specific kinesin, they do not act fast enough to distinguish between the direct and indirect consequences of a specific motor’s malfunction. Here we apply a novel chemical-genetic approach to engineer inhibitable KIF1A motors that function normally in the absence of a small, cell-permeable molecule but are then specifically and rapidly inhibited in the presence of the small molecule. Through this work, we have designed six inhibitable KIF1A motors, which we will further characterize through live cell and single molecule imaging. We also aim to use these motors to study the specific roles of kinesin-3 motors in axonal transport and neuronal disease. Furthermore, because of the kinesin motor domain’s high conservation, these results will be informative when designing inhibitable versions of other members of the kinesin superfamily.
Myosins 1291-Pos Board B359 Molecular Mechanism of Synchronous Force Generations among Myosin Molecules Motoshi Kaya1, Takumi Washio2, Hideo Higuchi1. 1 Dept. Physics, University of Tokyo, Tokyo, Japan, 2Frontier Science, University of Tokyo, Tokyo, Japan. For more than half a century, molecular mechanism of muscle contraction has been investigated by various experimental approaches. One of main questions is whether myosin molecules generate force cooperatively? To address the question, we have developed the experimental system, in which synthetic myofilaments interact with a single actin filament. Our findings suggest that myosin molecules may generate force synchronously against high loads. To gain insight into the mechanism of cooperative force generations, we developed the simulation model consisting of 17 myosin molecules arranged in series and interacting with a single actin filament. The model revealed that strain-dependent kinetics and multiple steps of power strike enhance a chance of synchronous force generations among myosin molecules. 1292-Pos Board B360 O-Myo! An O-Shaped Myosin Gliding Assay for Characterizing LongTerm Actin-Myosin Behaviors Rizal F. Hariadi1, Abhinav J. Appukutty2, Sivaraj Sivaramakrishnan3. 1 Department of Physics and the Biodesign Institute, Arizona State University, Tempe, AZ, USA, 2Department Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA, 3Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA. Nature has evolved molecular machines that are critical in cellular processes occurring over broad timescales, ranging from seconds to days. As an example, myosin in the human heart take ~10 steps per second and are collectively responsible for ~10 heart beats per day. Analogous to the engineering of macro-scale machines, the evolution of molecular machines is likely to be constrained by the requirements of high short-term performance and a long lifetime. Here, we developed a circular gliding assay (O-Myo) that utilizes engineered micron-scale DNA nanotube rings with defined geometrical arrangements of dimeric myosin VI, a model system for myosin motor. The O-Myo gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 myosin motors per ring) multiple times, once per ring revolution, providing a method of assessing the long-term dynamics of actin-myosin interactions. Actin filaments glide along the nanotube rings with high processivity for up to 5 full revolutions (40 mm total run length; 11 minutes), consistent with cooperative interactions between a single actin filament and multiple myosin motors. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 mm). As a model application of OMEGA, we then analyze motor-based mechanical influence on ‘‘stop-and-go’’ gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes. 1293-Pos Board B361 Changes in Myosin Crossbridge Cycle during Human Development Alice Ward Racca, Samantha Lynn, Michael A. Geeves. University of Kent, Canterbury, United Kingdom.