Studying Plant ER-PM Contact Site Localized Proteins Using Microscopy

As in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for studying EPCS structures and identifying possible EPCS-associated proteins. These include using artificially constructed reporters, GFP tagged protein expression followed by image analysis, and immunogold labelling at the ultrastructural level. In combination, these methods can be used to identify the location of putative EPCS proteins, which can aid in predicting their potential subcellular function.