Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca²⁺-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rim^V5-Znf but not in rim^HA-Znf In addition, rim^HA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIM^HA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.