Magrassi, R. | Search bioRxiv

bioRxiv2022

Strategies and experimental tips for optimized quantitative single-molecule studies of membrane and membrane-associated proteins

The study of stoichiometry and supra-molecular organization of membrane (and membrane-associated) proteins plays a key role in understanding membrane structure and function. Single-molecule localization techniques (SML), besides providing imaging at unprecedented resolution, also offer quantitative tools such as stepwise photobleaching (SP) experiments and quantitative single-molecule localization (qSMLM). SML is becoming widely present in imaging core facilities but addressing biological problems by molecular counting experiments still remains not straightforward since experimental approaches for sample preparation require particular attention. We will focus on the experimental aspects that may prevent successful quantitative SML experiments of membrane-associated proteins. Depending on the specific experiment, to avoid artifacts and to miscount, fine-tuning of the expression levels and proper staining procedures are required, as well as optimized protocols and controls for counting. The work aims to highlight the crucial aspects that must be faced when quantitative single-molecule experiments are performed, helping to match the gap between sample preparation and the application of quantitative fluorescence microscopy techniques.

biophysics↗

bioRxiv2018

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.

biophysics↗