PT - JOURNAL ARTICLE AU - Mrestani, Achmed AU - Dannhäuser, Sven AU - Pauli, Martin AU - Kollmannsberger, Philip AU - Hübsch, Martha AU - Morris, Lydia AU - Langenhan, Tobias AU - Heckmann, Manfred AU - Paul, Mila M TI - Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging AID - 10.26508/lsa.202302021 DP - 2023 Dec 01 TA - Life Science Alliance PG - e202302021 VI - 6 IP - 12 4099 - https://www.life-science-alliance.org/content/6/12/e202302021.short 4100 - https://www.life-science-alliance.org/content/6/12/e202302021.full SO - Life Sci. Alliance2023 Dec 01; 6 AB - 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 rimV5−Znf but not in rimHA−Znf. In addition, rimHA−Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA−Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.