Microtubule Defects Influence Kinesin-Based Transport In Vitro
📝 Abstract
Microtubules are protein polymers that form “molecular highways” for long-range 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 defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.
💡 Analysis
Microtubules are protein polymers that form “molecular highways” for long-range 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 defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.
📄 Content
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Microtubule defects influence kinesin-based transport in vitro
Winnie H. Liang,1 Qiaochu Li,1 K M R. Faysal,1 Stephen J. King,2 Ajay Gopinathan,1 and Jing Xu1, *
1Department of Physics, University of California, Merced, CA 95343 2Burnett School of Biomedical Sciences, University of Central Florida, FL 32827
*Correspondence: Jing Xu (jxu8@ucmerced.edu)
RUNNING TITLE: Microtubule defects impact kinesin function
KEYWORDS: Microtubule lattice, Molecular motors, Optical trapping, Taxol, Pause
ABSTRACT Microtubules are protein polymers that form “molecular highways” for long-range 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 defects impact molecular-motor based transport remains unknown. Here we report a new approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.
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INTRODUCTION Microtubules are biopolymers that self-assemble from tubulin dimers (1-5). During self- assembly, tubulin dimers stack longitudinally to form linear protofilaments, with multiple protofilaments associating laterally to form a hollow tubular structure, the microtubule (Fig. S1 A in the Supporting Material). Each microtubule is not necessarily perfect and can exhibit packing mistakes in the tubulin dimers (defects). The range of defects in microtubules include missing tubulin dimers (4, 5) and changes in the number of assembled protofilaments (1-3) (Fig. S1 B). These defects have been observed for microtubules in vitro (1-5) and in cell extracts (1). Molecular motors such as kinesin rely on microtubules as molecular highways to drive mechanical transport in cells (6-10). This transport is critical for eukaryotic cell function and survival. Each individual kinesin typically tracks a single protofilament in each microtubule (11). Since microtubule defects include disruptions within individual microtubule protofilaments (Fig. S1 B), we hypothesized that these defects may influence kinesin-based transport. A key experimental challenge for testing our hypothesis is that microtubule defects cannot be directly observed in current motility experiments using optical microscopes. Label-free imaging is not yet possible because the physical size of microtubule defects is ~1/20th below the optical resolution limit. There are also no known biomarkers for specific and non-invasive labeling/imaging of these structural defects, as the biochemical nature of the tubulin dimer within/surrounding these lattice defects is not yet understood. To overcome these experimental challenges, we developed a single-microtubule assay to probe the effects of microtubule defects on kinesin-based transport in vitro. Since molecular motors typically work in small groups to transport materials in cells (6-10), we focused our investigations on cargo transport by more than one kinesin. To address the effects on transport by different motor numbers, we examined two regimes: one in which each bead was carried by a few kinesins and one in which each bead was carried by many motors. We found that microtubule defects influence kinesin-based transport in a manner that depends on the number of motors present on the cargo.
MATERIALS AND METHODS Proteins and reagents Kinesin and tubulin were purified from bovine brains as previously described (12, 13). Kinesin, which was microtubule-affinity purified and free of “dead” motors (12), was flash frozen in PMEE buffer (35 mM piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), 5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, pH 6.8) supplemented with 45% glycerol and 1 mM dithiothreitol. Tubulin, which was free of microtubule-associated proteins (13), was flash frozen in PM buffer (100 mM PIPES, 1 mM MgSO4, 2 mM EGTA, pH 6.9) supplemented with 45% glycerol and 1 mM dithiothreitol. Anti-tubulin antibody (T7816, clone SAP.4G5), poly-L-lysine (P8920), Pluronic F-127 (P2443), and chemicals (unless otherwise specified) were purchased from Sigma- Aldrich (St. Louis, MO, USA). Dimethyldichlorosilane solution (2% wt/vol, Repel-Silane ES) was purchased from GE Healthcare Bio-Sciences (Marlborough, MA, USA). Guanaly
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