Quantifying motor protein copy number in super-resolution using an imaging invariant calibration

Motor proteins are nanoscale machines that convert the energy of ATP hydrolysis into the mechanical motion of walking along cytoskeletal filaments. In doing so, they transport organelles and help maintain sub-cellular organization. We previously developed a DNA origami-based calibration approach to extract protein copy number from super-resolution images. Using this approach, we show here that the retrograde motor protein dynein is mostly present as a single motor in the cytosol, whereas a small population of dynein along the microtubule cytoskeleton forms higher-order multimers organized into nano-sized domains. We further demonstrate, using dynein as a test sample, that the DNA origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any GFP-fused protein of interest. Our results have implications for motor coordination during intracellular trafficking as well as for using super-resolution as a quantitative method to determine protein copy number at the nanoscale level.