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Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

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Researchers precisely located the Rab-3-interacting molecule (RIM) protein within active zones at the Drosophila neuromuscular junction. This revealed how RIM

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Molecular Biology

Background:

  • Chemical synaptic transmission relies on neurotransmitter release from presynaptic active zones (AZs).
  • The Rab-3-interacting molecule (RIM) protein is crucial for normal calcium-triggered neurotransmitter release.
  • The exact localization of RIM within AZs at glutamatergic neuromuscular junctions in Drosophila melanogaster is not fully understood.

Purpose of the Study:

  • To determine the precise localization of the RIM protein within presynaptic active zones.
  • To investigate the functional impact of RIM tagging on synaptic transmission and plasticity.
  • To characterize the nanoscale organization of RIM within AZs using super-resolution microscopy.

Main Methods:

  • CRISPR/Cas9 genome engineering to create N-terminally tagged RIM variants (RIM-V5 and RIM-HA).
  • Super-resolution localization microscopy to visualize tagged RIM proteins.
  • Electrophysiological recordings to assess synaptic transmission and plasticity.
  • Hierarchical clustering analysis to quantify RIM subcluster organization.

Main Results:

  • Both V5 and HA tags were successfully integrated, and tagged RIM proteins co-localized with the Bruchpilot scaffold.
  • AP-evoked synaptic release was impaired in RIM-V5 but not in RIM-HA tagged flies.
  • RIM-HA synapses exhibited intact presynaptic homeostatic potentiation.
  • Super-resolution imaging revealed approximately 10 RIM-HA subclusters per AZ, with a diameter of ~13 nm.
  • These subclusters were found to be compacted and increased in number during presynaptic homeostatic potentiation.

Conclusions:

  • N-terminal tagging of RIM can differentially affect synaptic function, with the HA tag being more suitable for functional studies.
  • RIM is organized into nanoscale subclusters within the active zone.
  • The organization of RIM subclusters is dynamic and changes with presynaptic plasticity, suggesting a role in regulating synaptic efficacy.