Cytoplasmic Dynein Ring Tilting Detected by Combined polTIRF and Sub-Pixel Particle Tracking of Semiconductor Quantum Rods

Abstract

The mechanism of cytoplasmic dynein, a microtubule-based motor responsible for the majority of minus-end-directed transport in eukaryotes, remains poorly understood compared to myosin and kinesin. While recent crystal and EM structures have given insight into the conformational changes that occur in the AAA+ ring of dynein during ATP hydrolysis, dynamic structural information is lacking. In order to better understand the dynein translocation mechanism we combine polarized total internal reflection fluorescence (polTIRF) microscopy and high precision localization to simultaneously track position and orientation of single dynein molecules in real time. CdSe/CdS rod-in-rod nanoparticles with polarized emission are coated with mercaptoundecanoic acid (MUA) and functionalized with NeutrAvidin. The polarized nanorods are bifunctionally conjugated via biotin-avidin linkages to biotinylation sites inserted in two positions in dynein's AAA+ ring. Both homo- and hetero-dimeric dynein constructs are analyzed. Fluorescence emission of dynein-conjugated rods is split into four channels based on polarization and imaged with an EMCCD camera. The relative intensities of the four channels are used to determine the three-dimensional orientation of the rod, and therefore the dynein AAA+ ring, during stepping. Using this combined polTIRF/tracking method we detect tilting of the ring domain during stepping. Ring rotations are ATP-dependent, highly irregular and are mainly in the plane of the microtubule when correlated with translocation events. Supported by NIH grant P01GM087253.