Single Molecule Imaging of RIM1 Scaffold in Photoreceptor Neurotransmission

项目002 (079):RIM1支架在光感受器神经传递中的单分子成像

Project 002 (079): Single Molecule Imaging of RIM1 Scaffold in Photoreceptor Neurotransmission, Choi PL PROJECT SUMMARY/ABSTRACT Photoreceptors in the retina transmit a response to light across their synapses by regulating the release of glutamate-containing synaptic vesicles at the active zone. The photoreceptor synapse uses a ribbon-type active zone composed of multi-domain scaffold proteins to promote fast and continuous neurotransmitter release that is needed for normal vision. Dysregulation of the scaffold proteins involved in synaptic transmission is associated with retinal diseases leading to blindness. However, we have limited knowledge of the molecular mechanisms underlying these pathologies, which is critical for understanding and treating these diseases. Our long-term goal is to determine the molecular mechanisms of synaptic vesicle release at the active zone and how the cellular machinery is dysregulated in retinal disease leading to visual disabilities. The overall objective of this application is to determine how an autosomal dominant mutation in Rab3-interacting molecule 1 (RIM1) causes the blinding disease, cone-rod dystrophy type 7 (CORD7). Our central hypothesis for this proposal is that the structural dynamics of RIM1 regulates the scaffolding activity, which is disrupted by the CORD7 disease mutation (Aim 1), leading to impairment of a functional active zone by altering phase separation and vesicle fusion (Aim 2). RIM1 is the central component regulating the function of the active zone by directly or indirectly interacting with all other active zone proteins. RIM1 consists of tandem arrays of highly structured domains connected by intrinsically disordered linkers, which precludes structural analysis by traditional methods. To overcome this limitation, we will use the state-of-the-art single-molecule fluorescence resonance energy transfer (smFRET) to determine the dynamic structure of RIM1. We have established an in vitro reconstituted system recapitulating the synaptic geometry of the active zone, allowing protein dynamics and function to be observed in a biologically relevant context. Our novel assay will provide the first structural details of RIM1 and how the CORD7 mutation affects the function of RIM1 by altering the scaffolding activity. How the cellular machineries are organized to allow the release of synaptic vesicles is incompletely understood. RIM1 undergoes liquid-liquid phase separation (LLPS), condensing into a membrane-less “organelle”, when mixed with other active zone proteins. This scaffold organization suggests that LLPS is essential for vesicle release at the active zone. To determine the functional significance of RIM1 LLPS in vesicle fusion, we will conduct a novel single-vesicle fusion assay that we developed. These approaches will provide the first structural details of RIM1 during LLPS and how LLPS drives vesicle fusion. Achieving single-molecule resolution of protein dynamics and its function in a biological context related to blindness will uncover novel mechanisms of RIM1 that are hidden in ensemble measurements, and set a platform for molecular analysis of disease mutations such as CORD7.