Visualizing synaptic ribbons in the living cell.
Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains approximately 4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.
Zenisek
David
D
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. david.zenisek@yale.edu
Horst
Nicole K
NK
Merrifield
Christien
C
Sterling
Peter
P
Matthews
Gary
G
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000648:ultrastructure; D005136:Eye Proteins / Q000032:analysis; D005456:Fluorescent Dyes; D006054:Goldfish; D006624:Hippocampus / Q000166:cytology; D007700:Kinetics; D009475:Neurons, Afferent / Q000737:chemistry / Q000648:ultrastructure*; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure*; D010446:Peptide Fragments; D017729:Presynaptic Terminals / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D012160:Retina / Q000166:cytology; D013569:Synapses / Q000737:chemistry / Q000648:ultrastructure*; D013572:Synaptic Vesicles / Q000648:ultrastructure
D015220:Calcium Channels; C486016:Ctbp2 protein, rat; D005136:Eye Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments