Detecting torsional motion of kinesin motor proteins using birefringent microspheres and high-resolution optical tweezers

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Dokumentart: PhDThesis
Date: 2018-05-31
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Schäffer, Erik (Prof. Dr.)
Day of Oral Examination: 2017-05-05
DDC Classifikation: 500 - Natural sciences and mathematics
530 - Physics
570 - Life sciences; biology
Keywords: Kinesin , Biophysik , Optische Pinzette , Doppelbrechung
Other Keywords:
Molecular Biology
Optical Tweezers
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The kinesins motor proteins move cellular cargo on microtubule tracks. They are best known for their role in cell division and in axonal transport in neurons. A defect in kinesin function leads to diseases, typically involving defective transport of cell components or pathogens, or defects in cell division. The mechanics of kinesin motor motion has been studied extensively in the last decades. Because of the identical subunits, the motor has been proposed to rotate during stepping. For each step the motor is expected to rotate by 180-degree and a torque transferred from the motor head, through the stalk, onto the motor bound cargo should then be visible as angular steps for every translational step. Yet, experiments mostly done at low ATP concentrations only revealed occasional motor stalk reversals and an asymmetry of consecutive steps, so-called limping, which was attributed to loads perpendicular to the microtubule axis. The stepping rate of kinesin motors is slower at low ATP concentrations and faster at higher ATP concentrations. At high, physiological ATP concentration, rotations have not been detected because of long response times of rotational probes. Recent work on intermediate states during stepping indicate continuous, however, direct evidence for such rotational motion is lacking. Here, we used high-resolution optical tweezers combined with a sensitive optical micro-protractor and torsion balance employing highly birefringent, liquid crystalline probes to directly and simultaneously measure the translocation, rotation, and generation of force and torque of single kinesin-1 motors. Surprisingly, we found that motors translocating along microtubules at saturating ATP concentrations, rotated in a unidirectional manner, producing significant torques on the probes. Accounting for the rotational work, makes kinesin a highly efficient machine. Because motors also limped, these results imply that the motor's gait follows a rotary asymmetric hand-over-hand mechanism. Our method is generally applicable to study rotational motion of molecular machines and our findings have implications for kinesin-driven cellular processes. The results are consistent with a unidirectional rotation of kinesin motors in contrast to the current asymmetric hand-over-hand model which resembles a human gait. Sustained unidirectional rotation implies that, the motor head during stepping is not free to rotate. The detection and measurement of rotation using optical tweezers opens an additional dimension to the study of single bio-molecules.

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