Restoring visual function to blind mice with a photoswitch that exploits electrophysiological remodeling of retinal ganglion cells.

Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are blinding diseases caused by the degeneration of rods and cones, leaving the remainder of the visual system unable to respond to light. Here, we report a chemical photoswitch named DENAQ that restores retinal responses to white light of intensity similar to ordinary daylight. A single intraocular injection of DENAQ photosensitizes the blind retina for days, restoring electrophysiological and behavioral responses with no toxicity. Experiments on mouse strains with functional, nonfunctional, or degenerated rods and cones show that DENAQ is effective only in retinas with degenerated photoreceptors. DENAQ confers light sensitivity on a hyperpolarization-activated inward current that is enhanced in degenerated retina, enabling optical control of retinal ganglion cell firing. The acceptable light sensitivity, favorable spectral sensitivity, and selective targeting to diseased tissue make DENAQ a prime drug candidate for vision restoration in patients with end-stage RP and AMD.

Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. In the present study, we used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy between fluorescently labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly before endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only present in docked vesicles; unexpected residual "orphan SNARE complexes" also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis-conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatiotemporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid intermolecular interactions between small proportions of protein partners in cellular subcompartments.

Increased motion and travel, rather than stable docking, characterize the last moments before secretory granule fusion.

The state of secretory granules immediately before fusion with the plasma membrane is unknown, although the granules are generally assumed to be stably bound (docked). We had previously developed methods using total internal reflection fluorescence microscopy and image analysis to determine the position of chromaffin granules immediately adjacent to the plasma membrane with high precision, often to within approximately 10 nm, or <5% of the granule diameter (300 nm). These distances are of the dimensions of large proteins and are comparable with the unitary step sizes of molecular motors. Here we demonstrate with quantitative measures of granule travel in the plane parallel to the plasma membrane that secretory granules change position within several hundred milliseconds of nicotinic agonist-induced fusion. Furthermore, just before fusion, granules frequently move to areas that they have rarely visited. The movement of granules to new areas is most evident for granules that fuse later during the stimulus. The movement may increase the probability of productive interactions of the granule with the plasma membrane or may reflect the pull of molecular interactions between the granule and the plasma membrane that are part of the fusion process. Thus, instead of being stably docked before exocytosis, granules undergo molecular-scale motions and travel immediately preceding the fusion event.