Browsing by Subject "Kinesin"
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Item Analysis of mutations in the kinesin motor that decouple ATPase activity and microtubule interaction(2004) Auerbach, Scott David; Johnson, Kenneth AllenConventional kinesin is a dimeric, microtubule-dependent motor whose activity is tightly coupled to ATP hydrolysis. Mutations that might affect the coupling between ATPase and motor activities of kinesin were predicted to fall within the gamma- phosphate sensor apparatus, a set of domains in the protein believed to detect the phosphorylation state of the bound nucleotide and mechanically transmit the information via conformational change to the microtubule-binding domain. An additional element, the relay helix, has been postulated to undergo axial translation, rotation, and/or elongation, in response to the loss of the gamma phosphate from the bound nucleotide, and serve as intermediary between nucleotide- and microtubule-binding sites. An N-terminal truncation of rat conventional kinesin was examined using steady- and transient-state kinetic methods. Rate constants for ATP and microtubule binding were determined, as well as those for microtubule-dependent ADP and phosphate release. The dimeric state of the motor in solution was confirmed using analytical ultracentrifugation. Conserved residues within the gamma phosphate sensor were selected for mutagenesis. The residue E237 is believed to form a transient salt bridge with R204 when the motor is in the ATP state, based on crystal structure analysis, and the mutations E237A and E237D were examined using transient state kinetic methods. Both mutants showed > 10-fold reduction in steady-state ATPase activity, although rate constants for ATP and microtubule binding, as well as ADP release were little affected. These results suggested a disruption in the catalysis step caused by the mutations. An electrostatic interaction between E200 and R204 may also form in response to changes in nucleotide phosphorylation state, however, E200D and E200A mutants were scarcely compromised in steady-state ATPase activity, and this was attributed to a reduction in the rate constants governing product release. Finally, the N256K mutation caused a >1000- fold reduction in the rate of ADP release and a ~100-fold reduction in the steady-state ATPase rate. N256 falls within the relay helix, although the mechanism by which the N256K defect arises cannot yet be determined.Item Imaging molecular motor regulation at the single molecule level(2013-12) Walther, Juergen Herbert; Shubeita, George T.Molecular motor proteins are responsible for the long range transport of vesicles and organelles inside living cells. A small number of motor types transport thousands of distinct cargoes to various regions in the cell at the same time. This requires that intracellular transport be tightly regulated, yet the details of how motor regulators and cofactors tune motor function remain unknown in most cases. In-vitro studies at the single motor level have been instrumental in understanding the function of individual motors. In this thesis work I developed the methodology to extend in-vitro experiments to interrogate motor regulation at the single molecule level. I describe my modifications to the microscope setup as well as the acquisition cycle that made this possible. By combining differential interference contrast microscopy with single molecule fluorescence imaging and optical trapping I was able to manipulate and image the cargo while imaging a fluorescently-labeled regulator binding at the site of the motors. I used lipid droplets purified from Drosophila embryos as cargoes. Lipid droplets are carried by the opposite polarity microtubule motors kinesin and dynein in the embryos, and bind specifically to microtubules in-vitro. In the presence of ATP they exhibit long-range and short-range motility. For this proof-of-principle experiment I used fluorescently labeled AMPPNP, a non-hydrolysable analogue of ATP which binds to the motor domain of kinesin when microtubule-bound, to image the binding of the nucleotide to the motor and demonstrate the activity of the motors. While a large fraction of microtubule-bound droplets co-localized with a fluorescent AMPPNP molecule, non-specific binding of the nucleotide to the microscope slide surface prevented confirming the specificity of the colocalization events. Nevertheless, these data demonstrate the ability of the methodology to capture, in real time, the process of a regulator binding the motor at the single molecule level.Item Measuring molecular motor forces to probe transport regulation in vivo(2012-05) Leidel, Christina Paulette; Shubeita, George T.The cell relies on molecular motor proteins for long range transport of vesicles and organelles to maintain the organization required within the cell as it changes over time. Cargos move bidirectionally along microtubules due to the presence of multiple copies of opposite polarity motors. Individual motor properties have been teased out in vitro, but understanding how multiple motors cooperate in vivo has thus far been limited by many obstacles. The goal of this work is to study how multiple similar and dissimilar motors operate together in vivo. Since the function of motors is to generate force to haul cargos, I designed a novel optical trapping system capable of precisely measuring the forces exerted by molecular motors in their native environment, a living cell. Using this system, I find evidence that motors do not fight against each other, supporting the regulation model over the tug-of war model for bidirectional transport. I then study motor regulation in axons in the context of Alzheimer’s disease. I find that GSK-3, a kinase found in abnormal amounts in Alzheimer’s brains, is a negative regulator of transport. I show that GSK-3 regulates motor activity rather than cargo binding. Finally, I also use the optical trap to probe the viscosity of cytosol in vivo and investigate its implications on the cooperation of multiple motors.Item Probing the coupling mechanism of opposite polarity motors(2010-08) Holzmeister, Phil Jack; Shubeita, George T.; Florin, Ernst-LudwigMolecular motors are responsible for all long range transport and organization of organelles within cells. However, little is known about the interaction of multiple similar and dissimilar motors. In this thesis I describe experiments to probe the coordination of the motors kinesin and dynein which move towards the opposite ends of microtubules. Cargos they haul show bidirectional movement at short scales yet there is net transport in one direction or the other. Two distinct models for the bidirectional transport exist: regulation and a tug-of-war. In order to differentiate between them, kinesin-specific antibodies are injected into Drosophila embryos and the effect on transport of lipid droplets is quantified and compared to unperturbed motion. The function-blocking antibodies resulted in an increased run length of dynein-mediated transport and a decrease in that of kinesin. Furthermore, reduced velocities in both directions and a trend towards shorter pauses were observed. Comparison of these results to predictions the models provide for this scenario supports a tug-of-war model rather than regulation.Item Structural Basis for Coordination in Dimeric Kinesin(2009-09-04) Metlagel, Zoltan; Kikkawa, MasahideKinesin-1 (conventional kinesin) is a protein motor that carries organelles and vesicle cargo along its microtubule track. The two catalytic heads of Kinesin-1 are linked to function as a highly processive "molecular walker'' that can take hundreds of steps before falling off the track. A key requirement for processivity is that the nucleotide cycles of the heads are coordinated to prevent simultaneous release of both heads from the track. The structural basis for coordination has not been established yet. Here, we show the conformational changes involved in nucleotide-dependent switching of the kinesin core in the functional context of the \MT. The observed conformational differences between two key nucleotide states comprise the structural groundwork for future studies on how the nucleotide cycles are coordinated between the heads. Further, a software suite, Ruby-Helix, was developed to facilitate helical image analysis and implement a new algorithm for the analysis of helical objects with a seam. Ruby-Helix incorporates several new techniques for conventional helical analysis, and automates many of the repetitive steps involved in helical analysis, thereby greatly increasing the throughput of this method.