Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging
09/11/2023
Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging
Achmed Mrestani, Sven Dannhäuser, Martin Pauli, Philip Kollmannsberger, Martha Hübsch, Lydia Morris, Tobias Langenhan, Manfred Heckmann, Mila M Paul (2023)
life-science-alliance, vol 6, no 12, e202302021, 2023
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.
