Fig. 5

PS1 interacts with Syt1 in Ca²⁺-dependent manner in primary neurons. a Co-immunoprecipitation experiments demonstrate that KCl (n = 3) or calcium ionophore A23187 treatment (n = 3) of primary neurons that trigger Ca²⁺ influx strengthen the interaction between PS1 and Syt1. The asterisk shows a non-specific, heavy chain IgG band. The graph presents quantitative analysis of the Syt1 band intensity, mean ± SEM. Statistical significance was determined using the unpaired student t-test, ** p < 0.01, *** p < 0.001. b FLIM analysis of the PS1-Syt1 proximity in mouse cortical primary neurons treated for 5 minutes with 50 mM KCl (KCl+) or water control (KCl-). Fluorescence images show PS1 (green) and Syt1 (red) immunoreactivity. Scale bar: 5 μm. Pseudo-colored FLIM images depict lifetime of the Alexa 488 donor fluorophore. Colorimetric scale shows fluorescence lifetime in picoseconds. Zoomed boxed area shows FLIM image superimposed onto a table indicating average lifetimes for each pixel (~0.2 um²) of the image. Shortest lifetimes (yellow-to-red) reflect closest proximity between PS1 and Syt1. Bar graph presents [%] FRET efficiency (PS1-Syt1 proximity) recorded in the outlined regions of interest (ROIs) corresponding to the neuronal cell bodies or the processes. (mean ± SEM; n = 82 for cell bodies KCl(-), n = 63 for cell bodies KCl(+), n = 128 for processes KCl(-) and n = 146 for processes KCl(+); unpaired student t-test, *** p < 0.001). PS1 presenilin 1, Syt1 synaptotagmin 1, FLIM fluorescence lifetime imaging microscopy, FRET Förster Resonance Energy Transfer