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

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dc.contributor.advisor Schäffer, Erik (Prof. Dr.) Ramaiya, Avin 2017-06-02T06:15:21Z 2017-06-02T06:15:21Z 2018-05-31
dc.identifier.other 506173631 de_DE
dc.identifier.uri de_DE
dc.description.abstract 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. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podno de_DE
dc.rights.uri de_DE
dc.rights.uri en
dc.subject.classification Kinesin , Biophysik , Optische Pinzette , Doppelbrechung de_DE
dc.subject.ddc 500 de_DE
dc.subject.ddc 530 de_DE
dc.subject.ddc 570 de_DE
dc.subject.other Biophysics en
dc.subject.other Molecular Biology en
dc.subject.other Birefringence en
dc.subject.other Motility en
dc.subject.other Optical Tweezers en
dc.title Detecting torsional motion of kinesin motor proteins using birefringent microspheres and high-resolution optical tweezers en
dc.type Dissertation de_DE
dcterms.dateAccepted 2017-05-05
utue.publikation.fachbereich Biologie de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE


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