Direct Observation of the Allosteric Conformational Change of Kinesin-1 using Gold Nanorod and its Implication for Head-Head Coordination

Monday, February 29, 2016 Our data contribute to the mechanochemical dissection of this divergent mitotic kinesin and emphasize the critical mechanistic contribution of the motor interface with the microtubule. 961-Plat Kinetics of Nucleotide-Dependent Structural Transitions in the Kinesin-1 Hydrolysis Cycle Keith J. Mickolajczyk1, Nathan C. Deffenbaugh1, Jaime Ortega-Arroyo2, Joanna Andrecka2, Philipp Kukura2, William O. Hancock1. 1 Biomedical Engineering, Penn State University, University Park, PA, USA, 2 Physical and Theoretical Chemistry, Oxford Univerity, Oxford, United Kingdom. To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. The high spatiotemporal resolution of this method enabled real-time recording of structural changes in the protein as it walked at ~100 steps per second. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying the structural transitions to and from this one-headbound intermediate were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, meaning that there is one rate-limited step in each the one- and two-heads bound states. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor, and show that the mechanism underlying stepping is a two-step process. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily. 962-Plat Direct Observation of the Allosteric Conformational Change of Kinesin-1 using Gold Nanorod and its Implication for Head-Head Coordination Yamato Niitani1, Sawako Enoki2, Hiroyuki Noji2, Ryota Iino3, Michio Tomishige1. 1 Department of Applied Physics, The University of Tokyo, Tokyo, Japan, 2 Department of Applied Chemistry, The University of Tokyo, Tokyo, Japan, 3 Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, Okazaki, Japan. Kinesin-1 is a motor protein that moves along microtubules by alternately moving two motor domains (heads) in a hand-over-hand manner. The neck linker, short stretch that connects two heads, has been shown to essential for the coordination between heads, although the underlying structural basis is unknown. Recently solved crystal structure revealed a subdomain rotation that takes place upon ATP hydrolysis; closing the nucleotide-binding pocket and opening a hydrophobic pocket at the opposite side, which will be filled with the neck linker. We hypothesized that this subdomain rotation is required for promoting ATP hydrolysis but is suppressed in the leading head because backward tension prohibits the docking of the neck linker. Here we tested this hypothesis by directly observing the rotational motion of the subdomain of one of kinesin heads during processive movement. We observed gold nanorods specifically attached to one of the heads using dark-field microscopy and determined its angles and positions at 100 ms temporal resolution and one-degree angle precision (Enoki et al. Anal. Chem. 2015). The angle of gold nanorod changed once while the labeled head binds to the microtubule and the angle reversed after detachment from microtubule. The dwell times in the pre- and post-rotation states were nearly equal at saturating ATP condition, indicating that the labeled head is in the pre- or post-rotation states when the head is in the leading or trailing positions, respectively. Mutation in the neck linker, which destabilizes the necklinker docking, increased the dwell time in the pre-rotation state, supporting the idea that the neck-linker docking is essential for stabilizing the subdomain rotation. These results explain how the neck linker tension/orientation allosterically regulates ATP hydrolysis differently in the leading and trailing heads. 963-Plat Impacts of Microtubule Structural Defects on Kinesin-Based Transport Winnie H. Liang1, Qiaochu Li1, K. Faysal1, Stephen J. King2, Ajay Gopinathan1, Jing Xu1. 1 Physics Graduate Group, University of California, Merced, Merced, CA, USA, 2Burnett School of Biomedical Sciences, University of Central Florida, Florida, FL, USA.

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Microtubules are protein polymers that form ‘‘molecular highways’’ for longrange transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these structural defects impact molecular-motor based transport remains unknown. Here we report a new approach that allowed us to directly investigate the impact of such structural defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargoes along individual microtubules in vitro. We found that structural defects in microtubules are a determining factor in kinesinbased transport. This impact depends on motor number: cargoes driven by two motors tended to dissociate prematurely from the microtubule, whereas cargoes driven by more motors tended to pause. Our study provides the first direct link between structural defects in microtubules and kinesin function, and suggests how alterations in cargo-motor number nontrivially tune this effect. Our study highlights the potential of kinesins as non-invasive biomarkers of structural defects in microtubules, and raises the possibility of microtubule defects as a previously unexplored mechanism for regulating kinesin-based transport in cells. 964-Plat Engineering Novel Actin-Based Molecular Motors from the MicrotubuleBased Motor Dynein Akane Furuta, Kazuhiro Oiwa, Hiroaki Kojima, Ken’ya Furuta. National Institute of Information and Communications Technology, Hyogo, Japan. Linear biomolecular motors such as myosin, kinesin and dynein are protein machines responsible for directional movement in the cell. Despite the extensive analyses that have been performed on these machines, the design and construction of a novel biomolecular motor still poses a formidable challenge. Here we adopt a bottom-up approach in which the existing protein modules from different cytoskeletal systems are combined to create new biomolecular motors. We show that the hybrid motors—combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins (ABPs)—robustly drive the sliding movement of an actin filament. The filament-binding affinity of the hybrid motors was nucleotide dependent, but the dependence was reversed when compared to that of the original dynein. Moreover, the direction of actin movement was able to be reversed simply by changing the relative position of the motor core and the actin binding module. Our synthetic strategy will open a way to understanding the design principle of biomolecular motors and thus to manufacturing controllable biomachines that work, for example, along artificial tracks at nanometer dimensions. 965-Plat Two Levels of Myosin-IIA Dynamics in Cells: Turnover of Filaments and Self-Organization of Filament Stacks Shiqiong Hu1, Kinjal Dasbiswas2, Zhenhuan Guo1, Yee-Han Tee1, Visalatchi Thiagarajan1, Ronen Zaidel-Bar1, Pascal Hersen3, Samuel Safran2, Alexander D. Bershadsky1,4. 1 Mechanobiology Institute, Singapore, Singapore, 2Departments of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel, 3Laboratoire Matie`re et Syste`mes Complexes, Universite´ Paris Diderot, Paris, France, 4 Departments of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Dynamics and self-organization of myosin-II filaments in non-muscle cells are poorly understood. Here, using structured illumination microscopy (SIM), we visualized with high resolution bipolar myosin-II filaments in REF52 fibroblasts by marking myosin-II light chain (MLC), or myosin-IIA heavy chain (MHC-IIA). The filaments demonstrated a uniform length of 300 nm and formed characteristic periodic striations along actin filament bundles, such as ventral stress-fibers, or transverse arcs in spreading cells. Myosin IIA striations were alternating with domains of variable length enriched by actin cross-linking protein alpha-actinin. Free barbed ends of actin filaments (visualized by incorporation of labeled G-actin) were localized between myosin-II striations, while pointed ends of actin filaments (visualized by labeling tropomodulin-3) were enriched in the regions corresponding to myosin-II striations. The most striking feature of the myosin-IIA filament organization was in registry alignment of the myosin-IIA filaments with subsequent formation of several microns long filament ‘‘stacks’’ that apparently formed bridges between parallel stress-fibers or arcs. FRAP experiments were performed in SIM showed that the recovery time for both GFP-MLC and GFP-MHC-IIA in tens of seconds and did not depend on actin filament turnover. However, the process of self-organization of the individual