Abstract 2935 Image Analysis of Gap Junction Plaque features and dynamics

Gap junctions (GJs) are formed by connexins (Cxs), membrane proteins that create channels and hemichannels, facilitating intercellular communication and the exchange of ions, small metabolites, and signaling molecules (Ceriani & Mammano, 2013).These gap junction channels aggregate into plaques at the plasma membrane, where they are dynamically regulated: opened, closed, shuffled, and internalized (Koval et al., 2014).Fluorescently tagged Cxs expressed in adherent cells are commonly used to study GJs.However, overexpression in certain cell types, such as HEK293FT cells, can lead to the accumulation of fluorescent Cxs at various intracellular locations, complicating the visual evaluation and characterization of GJ plaques (Dunn et al., 2011).The goal of this work was to develop an image-based workflow to isolate and characterize gap junction plaques using a plasma membrane imaging mask established with the CellBrite membrane stain.To this end, HEK293FT cells were transiently transfected with GFP-tagged Cxs, fixed with methanol/acetone, and stained with Cell-Brite555.Confocal images were acquired, and Imaris software was used to create a mask that identifies the plasma membrane.GFP fluorescence that colocalized with the plasma membrane stain was visually consistent with GJ plaque structures (i.e., linear sheet-like arrangements) and could be characterized (e.g., area, number, etc.) (Lachmanovich et al., 2003).We present proof-of-concept data demonstrating that this technique can generate metrics for comparing the characteristics of GJ plaques formed by different Cx proteins, evaluating the trafficking of wild-type and mutant Cx proteins, and assessing the dynamics of multiple co-expressed isoforms within plaques (Shelby et al., 2023).This work lays the foundation for a deeper understanding of gap junction biology and its implications for health and disease.