Methodological Nuances of Measuring Membrane Protein Nanoscopic Organization: A Case of Dopamine Transporter

Abstract

Super-resolution microscopy is an umbrella term for a family of powerful imaging techniques that examine the organization and dynamics of biomolecules at the nanoscale. However, an increasing number of super-resolution techniques along with their distinct limitations make it a nontrivial task to select the right technique to answer a specific question. Moreover, effective integration and interpretation of the multimodal, often conflicting data present another unique challenge, underscoring the need for experimental validation of super-resolved membrane protein organization. To shed light on the discrepancy between different super-resolution techniques, the impact of dopamine transporter labeling strategy and cell processing on its dynamic behavior was examined in catecholaminergic CAD cells. To this end, dopamine transporter fused to yellow fluorescent protein at the amino terminus was expressed in CAD cells and externally labeled with antagonist-conjugated quantum dot probes. The dual labeling strategy allowed direct comparison of transporter nanoscopic organization in live and fixed cells obtained via either yellow fluorescent protein tracking or quantum dot tracking in the same field of view. Our data indicated that the labeling strategy along with fixation had a significant impact on observed transporter surface dynamics and highlighted the importance of careful experimental vetting of the results obtained via super-resolution imaging.