Figure S1

We examined whether BDNF and MMP-9 are stored in dendritic spines, positioning them for rapid engagement in synaptic plasticity. To achieve this, we created a fusion protein by attaching pH-sensitive green fluorescent protein (superecliptic pHluorin, SEP) to a C-terminus of MMP-9. For comparison with BDNF, we used the previously described BDNF-SEP construct. We transfected rat hippocampal neuronal cell cultures with plasmids encoding tdTomato (for visualizing dendritic spine morphology), together with plasmids for either MMP-9-SEP or BDNF-SEP. We have used immunostaining to image MMP-9 and BDNF-containing vesicles. To improve the final resolution of the imaged neurons, we employed the expansion microscopy technique (expanded 4-fold, as measured by nuclei signal). We then imaged these samples using confocal microscopy, and images of dendritic spines were used for the reconstruction of vesicles and cell morphology using Imaris Image Analysis Software. We confirmed the storage of both BDNF- and MMP-9-containing vesicles within dendritic spines. BDNF-containing vesicles had an average diameter of 149.6 ± 2.4 nm, whereas those containing MMP-9 had an average diameter of 135.9 ± 2.6 nm. The data contain mean vesicle diameter in all measured cells as well as calculated expansion ratio based on Hoechst-stained nuclei.