Fig. 1: Visualizing astrocyte gap junctional communication using the astrocyte network tracer.
From: Astrocytes connect specific brain regions through plastic networks
a, Diagram of the astrocyte network tracer construct. b, Cx43 connexons contain six connexins, only one of which needs to be the fusion protein for TID to reside on the gap junction. c, Infected astrocytes biotinylate molecules that flux through Cx43 gap junctions into adjacent, uninfected cells. d, This volume-fills in-network astrocytes with biotinylated molecules such that infected (HA+streptavidin+), in-network (HA−streptavidin+) and out-of-network (HA−streptavidin−) cells can be visualized. e, Infection of primary immunopanned rat astrocytes (GFAP, green) resulted in Cx43–TID–HA expression (cyan) in 10% of cells, forming a biotin-labelled network containing 80% of cells (streptavidin, magenta) (Extended Data Figs. 1 and 2). f, Primary rat astrocytes were expanded 8.7× to visualize individual Cx43 gap junctions (grey) and incorporated fusion protein (HA, cyan). g, Super-resolution microscopy analysis of expanded astrocytes20 enabled single-molecule imaging of the fusion protein incorporated into astrocyte gap junctions expressed in immunopanned primary rat astrocytes. Polyclonal Cx43 antibodies conjugated directly to fluorophores reveal gap junction structure (grey), and the HA tag (cyan) shows TID within the gap junction vestibule of a small gap junction plaque. h, Single-molecule localization microscopy (SMLM) analysis of HA (cyan) and Cx43 (magenta). i, Quantification of the intrafluorophoric distance from HA to the nearest Cx43. The median (20.73 Å) is indicated by a solid blue line; the dotted lines denote the 25th (18.09 Å) and 75th (25.74 Å) percentiles. j, In vivo experimental timeline. Three weeks after viral injection, mice received biotin-supplemented drinking water for 1 week, after which they were perfused. The brains were then delipidated, stained with streptavidin to reveal biotinylated molecules, cleared and imaged using light-sheet microscopy. k, Light-sheet micrograph of the HA-tag (cyan) shows gap junctions containing fusion protein in an infected cell within a cleared brain. l, Streptavidin (magenta) labelling of biotinylated molecules. m, Merged image showing a two-cell example of an infected (left) and an uninfected in-network (right) cell. Scale bars, 20 μm (k–m); 100 μm (e); 23 μm, expanded to 200 μm (f); 574 Å, expanded to 500 nm (g); and 114 Å, expanded to 100 nm (h).
