Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation
Figure 3
BLINC for imaging neurexin-neuroligin interactions in neuron cultures and in HEK-neuron mixed cultures.
(A) BLINC labeling of pure neuron cultures. Two pools of hippocampal neurons were separately nucleofected at DIV0 with BirA-NRX plus a membrane tdTomato marker (shown in blue), or 3xAP-NLG1 plus a Venus marker (shown in green). For the top row, the BirA36-NRX3β construct was used, and for the bottom row the BirA272-NRX3β construct was used. All constructs had CAG promoters. Labeling was performed at DIV5 with biotin+ATP for 15 minutes, followed by monovalent streptavidin-AF647 detection for 5 minutes. Confocal images of live neurons showed no detectable BLINC signal for the BirA36-NRX3β fusion across 10 fields of view in which Venus- and Tomato-expressing neurons were observed to be crossing. For the BirA272-NRX3β fusion (bottom row), BLINC signal was detected in 5 out of 10 such fields of view. (B) BLINC labeling of mixed HEK-neuron cultures. HEK cells expressing BirA272-NRX3β and a dsRed marker (shown in blue) were plated on top of rat hippocampal neurons transfected with lipofectamine at DIV10 with 3xAP-NLG1 plus a Venus marker (shown in green). Labeling was performed at DIV11 as in (A). BLINC signal could be detected in 22 out of 30 fields of view, and was localized to contact sites (arrow heads). The bottom row shows a control with a D137A mutation in NRX3β; BLINC signal was not observed in any field of view. All scale bars, 10 µm.