Sebastian Markert (Würzburg / DE), Timothy Mulligan (Baltimore, MD / US), Liyun Zhang (Baltimore, MD / US), James Thierer (Baltimore, MD / US), Jeff Mumm (Baltimore, MD / US), Shigeki Watanabe (Baltimore, MD / US)
Abstract text (incl. figure legends and references)
Our eyes offer us a window into the world. This is made possible first and foremost by the photoreceptor neurons in our retinas. These neurons perceive light and translate it into electrical nerve signals that our brain can understand and process. This translation from light to nerve signals is achieved at so-called ribbon synapses. There, neurotransmitter is released from vesicles that are lined up at ribbon structures. The amount of transmitter release over time is inversely correlated with light intensity: more light, less transmitter. In the dark, the rate of release is at maximum. In particular, right at light-dark transition, a huge amount of transmitter is released within milliseconds. It is unknown how such bursts of release can be achieved in such a short amount of time. The field has been arguing about possible models for decades. The process is too fast, and vesicles are too small to visualize by live-cell fluorescence microscopy. However, with a new technique called flash-and-freeze, this question can finally be answered.
The flash-and-freeze technique is uniquely able to visualize cellular processes with extremely high spatial and temporal resolution. Retinas of adult zebrafish were subjected to light-dark transitions to elicit the transmitter bursts. During the bursts, the retinas were frozen under high pressure within milliseconds. This halts the cellular processes that produce the bursts, thus enabling us to examine these extremely fast events. We then use electron microscopy to visualize the ribbon synapses with nanometer-resolution.
Our data show unusually large vesicles at synaptic ribbons several milliseconds after light-off. Fusion events where sometimes captured in action. At later time points, these large vesicles are not observed anymore. In light-on controls, all vesicles remain their usual size.
Our data strongly suggest that vesicles undergo so-called compound fusion: They are lined up at the ribbon to fuse with each other to form super-vesicles. These giant vesicles then release a huge amount of transmitter at once, thus explaining the bursts. We are now investigating the molecular mechanisms of compound fusion.
The flash-and-freeze technique can be adapted to investigate other fast cellular processes at extremely high spatial and temporal resolutions.