Fig. 1

RIM1 is localized both presynaptically and postsynaptically. a Subcellular fractions of mouse cortex by synaptosome fractionation were probed for the presynaptic marker synaptophysin, the postsynaptic marker PSD-95, the RIM proteins RIM1 and RIM2, the RIM-related protein RBP2, the NMDAR subunits GluN2A and GluN2B, the AMPAR subunit GluA1, and other important synaptic proteins such as synapsin II, Rab3, and Rab5. H homogenates, S2 supernatant after first high-speed centrifugation (12,000×g), P2 pellet after the first high-speed centrifugation (12,000×g). b Subcellular fractions of mouse cortex by sucrose gradient centrifugation were probed for synaptophysin, PSD-95, RIM1, RIM2, RBP2, GluN2A, GluN2B, GluA1, and other important synaptic proteins such as synapsin II, Rab3, and Rab5. Total, homogenates; SPM synaptic plasma membrane fraction; PSD postsynaptic density fraction. c Subcellular fractions of mouse cortex by sucrose gradient centrifugation were probed for the presynaptic markers synaptophysin, Munc13, and Bassoon, the postsynaptic marker PSD-95, and RIM1. Total, homogenates; SPM synaptic plasma membrane fraction; PSD postsynaptic density fraction; 1F and 2F are represented as the normal and the 2-fold quantity of the PSD fraction sample, respectively. d Electron micrographs of adult mouse hippocampus stained for RIM1 with immunogold (data from 4 mice). Left: a nanogold-labeled axon terminal makes a synapse with an immunonegative dendritic spine. Middle: an immunonegative axon makes a synapse with a nanogold-labeled dendrite. Right: a nanogold-labeled axon and an immunonegative axon make synapses with a nanogold-labeled dendrite; a axon, d dendrite; blue arrow, synapse with nanogold in the presynaptic profile; red arrow, synapse with nanogold in the postsynaptic profile; green arrow, synapse with nanogold in both the pre- and postsynaptic profiles. All arrows point to the PSD. Scale bar, 200 nm