Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Thursday Speaker Abstracts

TRACKING DYNEIN STEPPING ALONG MICROTUBULES WITH NANOMETER ACCURACY USING THREE-COLOR IMAGING Stefan Niekamp 1 ; Nico Stuurman 1,2 ; Ronald D Vale 1,2 ; 1 University of California, San Francisco, Department of Cellular and Molecular Pharmacology, San Francisco, CA, USA 2 Howard Hughes Medical Institute, San Francisco, CA, USA Cytoplasmic dynein is a minus-end directed microtubule-based motor that belongs to the AAA family of proteins and is responsible for the transportation of many cargos in cells and plays a key role in mitosis. Unlike the well-known kinesin or myosin motor domains, which are globular and compact, the dynein motor domain contains a small microtubule-binding domain (MTBD) that is spatially separated by a ~135 A long coiled-coil from its large catalytic AAA ring. From previous work, in which the AAA rings of a dimeric dynein were labeled with fluorescent probes, it is known that dynein moves through uncoordinated stepping of the AAA ring domains. However, recent structural studies have shown that the relative orientation of the AAA ring and MTBD is quite flexible. Thus, to fully understand how dynein is walking, it is inevitable to follow the MTBD. Moreover, how different domains of dynein move relative to each other during every step cycle is unknown. Addressing these questions requires high-resolution, multicolor imaging. We therefore developed new methods for three-color image registration and distance measurements that enable us to determine distances between three colors with sub- nanometer accuracy. Moreover, we designed small fluorescent probes that allow us to track dynein about fifteen times longer than with common fluorescent dyes. With these new tools at hand we are beginning to see new patterns in dynein motility emerging. Together, we have developed new methods for three-color imaging with nanometer accuracy that provide insights into the mechanism of dynein stepping.

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