Resolving the molecular architecture of the photoreceptor active zone by MINFLUX nanoscopy

Abstract

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology indicate that neurotransmission depends on such optimization strategies, yet the molecular topography of the presynaptic Active Zone (AZ) where transmitter is released upon synaptic vesicle (SV) fusion remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. To facilitate MINFLUX nanoscopy of the AZ, we developed and verified an immobilization technique, we name Heat Assisted Rapid Dehydration (HARD). Here fresh retinal slices are directly stamped onto glass coverslips yielding a single layer of rod AZs. These AZs exhibited excellent labeling efficiency and minimal signal redundancy in the Z-direction. Our data indicate that the SV release site is a molecular complex of bassoon-Rab3-binding molecule 2 (RIM2)-ubMunc13-2-CAST. The complexes are serially duplicated longitudinally, and reflected in register along the axis of symmetry of the synaptic ribbon.

Digging Deeper: Advancements in Visualization of Inhibitory Synapses in Neurodegenerative Disorders

“…A newly developed combination of STED and STORM (MINFLUX, Abberior, company founded by Stefan Hell, https://abberior-instruments.com/knowledge/publications/, accessed on 17 November 2021) combines single-molecule activation with imaging using a super-fast STED-microscope to triangulate the exact position of the fluorophore. These results are in very high resolution (2 nm) and the imaging speed is faster compared with STORM [114,116].…”

“…Paasila et al [114] visualized the interactions between presynaptic terminals and microglia in situ, using dSTORM. The procedure they described opens the spectrum of molecular imaging using antibodies and SR microscopy to the analysis of routine formalin-fixed paraffin sections of the archival human brain.…”