Quantifying the Organization and Dynamics of the Plant Plasma Membrane Across Scales Using Light Microscopy

The plant cell surface continuum is composed of the cell wall, plasma membrane, and cytoskeleton. Plasmodesmata are specialized channels in the cell wall allowing intercellular communication and resource distribution. Proteins within these organelles play fundamental roles in development, perception of the external environment, and resource acquisition. Therefore, an understanding of protein dynamics and organization within the membrane and plasmodesmata is of fundamental importance to understanding both how plants develop as well as perceive the myriad of external stimuli they experience and initiate appropriate downstream responses. In this chapter, I will describe protocols for quantifying the dynamics and organization of the plasma membrane and plasmodesmata proteins across scales. The protocols described below allow researchers to determine bulk protein mobility within the membrane using fluorescence recovery after photobleaching (FRAP), imaging, and quantification of nanodomain size (with Airyscan confocal microscopy) and determining the dynamics of these nanodomains at the single particle level using total internal reflection (TIRF) single particle imaging.