ABSTRACT
Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).
ABSTRACT
Brassinosteroids (BR) are key hormonal regulators of plant development. However, whereas the individual components of BR perception and signaling are well characterized experimentally, the question of how they can act and whether they are sufficient to carry out the critical function of cellular elongation remains open. Here, we combined computational modeling with quantitative cell physiology to understand the dynamics of the plasma membrane (PM)-localized BR response pathway during the initiation of cellular responses in the epidermis of the Arabidopsis root tip that are be linked to cell elongation. The model, consisting of ordinary differential equations, comprises the BR-induced hyperpolarization of the PM, the acidification of the apoplast and subsequent cell wall swelling. We demonstrate that the competence of the root epidermal cells for the BR response predominantly depends on the amount and activity of H+-ATPases in the PM. The model further predicts that an influx of cations is required to compensate for the shift of positive charges caused by the apoplastic acidification. A potassium channel was subsequently identified and experimentally characterized, fulfilling this function. Thus, we established the landscape of components and parameters for physiological processes potentially linked to cell elongation, a central process in plant development.
