Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells.

Abstract

Membrane contact sites between organelles are essential for maintaining cellular homeostasis, which requires the continuous exchange of signaling molecules, ions, nutrients and lipids. Alterations of different contact sites are associated with a wide spectrum of human diseases. However, visualizing and quantifying these contact sites remains a challenge. This protocol describes the use of split-GFP-based contact site sensors (SPLICS) in microscopy applications for mapping organelle contact sites both in fixed and living cells. SPLICS sensors are engineered to express equimolar amounts of the organelle-targeted nonfluorescent β11 and GFP1-10 portions of the split-GFP protein in a single vector, and are capable of reconstituting fluorescence when two opposing membranes come into proximity. Reconstitution will occur only over the cell volume at defined contact sites resulting in a bright signal that can be detected easily and quantified automatically with specific custom-made plugins. The use of minimal targeting sequences facilitates targeting specificity and membrane coverage, avoiding artifacts due to full-length fusion protein overexpression and, thus, possible perturbations of the cell's physiology. SPLICS sensors engineered to simultaneously detect multiple contact sites within the same cell have been generated by exploiting the ability of the β11 GFP fragment to reconstitute different color-shifted variants of the GFP1-10 fragment. Here, we describe a detailed protocol to set up SPLICS expression in living cells (2-3 d), detection and acquisition (1 d), and automated quantification with custom plugins (1-2 d). We also advise on construct design and characterization for novel organelle contacts.