Macromolecular Architecture Of The Synapse

突触的大分子结构

• 批准号: 10263019
• 负责人: Thomas S Reese
• 金额: $ 109.52万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10263019
• 关键词: 3-Dimensional; A kinase anchoring protein; AMPA Receptors; Affinity; Animals; Antibodies; Architecture; Area; Behavior; Behavior Control; Binding; Binding Sites; Brain; CRISPR/Cas technology; Calcineurin; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colorado; Complex; Cyclic AMP-Dependent Protein Kinases; Development; Diffuse; Diffusion; Distal; Electron Microscopy; Electrons; Electrophysiology (science); Evolution; Family; Filament; Force of Gravity; Freeze Substitution; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Image; Individual; Information Storage; Knock-in; Knock-out; Label; Laboratories; Length; Light; Lighting; Location; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Membrane Proteins; Memory; Methods; Microscopic; Microtomy; Molecular; Molecular Conformation; Molecular Machines; Molecular Probes; Morphologic artifacts; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; National Institute of Neurological Disorders and Stroke; Nervous system structure; Neurons; Neurophysiology - biologic function; Optics; Oxides; Peptide Signal Sequences; Peripheral; Phosphotransferases; Polymers; Positioning Attribute; Postsynaptic Membrane; Primates; Process; Protein Array; Proteins; RNA Interference; Research; Resolution; Role; Scaffolding Protein; Scanning; Signal Pathway; Signal Transduction; Singlet Oxygen; Site; Stains; Structural Models; Structure; Sucrose; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic plasticity; System; Techniques; Thick; Tomogram; Universities; Vertebral column; Visualization; Work; calmodulin-dependent protein kinase II; density; functional loss; improved; information processing; innovation; insight; interest; knock-down; light microscopy; overexpression; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor; reconstruction; scaffold; structured data; synaptic function; tomography; trafficking

The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that is known to be a key site of memory, information processing and storage. In order to explore the detailed molecular organization of the PSD, we developed a method to freeze-substitute hippocampal cultures and then examine them in thin sections by EM tomography to show individual protein complexes in close to their natural setting within the PSD. The initial work employing tomography revealed that the core of the PSD is an array of membrane associated, vertically oriented filaments that contain the scaffold protein, PSD-95, in an extended configuration and a polarized orientation, with its N-terminus positioned at the postsynaptic membrane. This finding provided insight into the overall organization of the core structure of the PSD because scaffolding proteins, such as PSD-95 family MAGUK proteins, have distinct multiple, diverse binding sites for other proteins arrayed along their length. Thus, the regular arrays of PSD-95 impose an ordering on many other PSD proteins, including the glutamate receptors, and provide an overall plan for the structure of the PSD. We have used antibodies to label proteins in tomograms, but the antibody complexes appearing as filamentous structures in tomograms confounds the identification of the target protein. An indirect approach combining knockdown with tomography has proved quite some insights, but may be limited for identifying individual components of protein complexes in tomograms. We attempted to develop an alternative method for identifying the proteins using an expressible probe, miniSOG, which generates singlet oxygen upon blue light illumination, and then oxidizes diaminobenzidine (DAB) to form electron dense polymers visualizable by EM in the proximity of the tagged molecule. We overexpressed miniSOG-PSD-95 in neurons and found that the staining generated by miniSOG is too diffuse to localize molecules with better than 20 nm precision. We therefore used various dilutions of sucrose solution in the photoconversion process to slowdown molecular diffusion. Eventually, with the help of EM tomography, we found evidence that electron dense structures developed at the distal ends of some of the membrane associated vertical filaments known to contain PSD-95, thus allowed identification of miniSOG tagged PSD-95. Now we succeeded in making CaMKII-APEX2 construct to show individual CaMKIIs can be visualized with little diffusion, making it a very promising approach to label and identify molecules in neurons. The idea that the PSD-95 dependent scaffold stabilizes the PSD has been explored by using EM tomography to determine the effects of RNAi knock down of MAGUKs. We examined the effects of knocking down, simultaneously, three major MAGUK proteins: PSD-95, PSD-93 and SAP102, and EM tomography revealed significant loss from the central core of the PSD, including NMDA receptor structures, vertical filaments, and AMPA receptors. Electrophysiology measurements by collaborators from the Roger Nicoll laboratory characterizing the effects of the same knock down show significant functional loss of NMDAR and AMAPR type EPSCs at levels compatible with the structural losses. Electron microscopy also showed depletion of vertical filaments along with AMPAR type structures at the peripheral region of the PSD, and significant reduction in size of NMDAR clusters in the center of the PSD. These structural data indicate that vertical filaments corresponding to MAGUKs anchor AMPARs and are also a factor in organizing NMDARs. Thus, PSD-95 MAGUKs are demonstrated to be the essential organizer of glutamate receptors at the PSD. We also developed another line of research recently with the Rumbaugh Lab to characterize structural role of SynGAP, which negatively regulates AMPAR binding to the PDZ domain of PAS-95 MAGUKs at the PSD, we have used immunoEM to map the location, orientation and conformation of SynGAP at the PSD to arrive at a structural model on how SynGAP might regulate and control synaptic excitability. We are also in the process of identifying NMDARs directly in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab to knockout the obligated GluN1 subunit of NMDARs and reconstruction of the postsynaptic density (PSD) with dark field scanning EM tomography. We now have evidence confirming that the class of transmembrane structures containing large globular cytoplasmic profiles contain NMDARs, whereas the structures with smaller and flat cytoplasmic profile contain AMPARs. We are completing with M. DellAcqua, University of Colorado, study of the conformations and distribution of A Kinase Anchoring Proteins (AKAPs) hippocampal synapses. These molecules are membrane associated proteins known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA, PKC) and calcineurin, important for synaptic plasticity (LTP and LTD). This work is showing that there is a conformational change in AKAPs in the PSD, different from that at the extra synaptic membrane. This distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. In collaboration with the Roger Nicoll Lab, we are studying the effects of overexpressing constitutively activated CaMKII on synaptic structure and function. To ameliorate potential artifacts due to overexpressing of CaMKII, we are also using a newly developed CaMKII CRISPR knock-in construct which allow express and localization of endogenous CaMKIIs in neurons. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM or thick section STEM tomography. Finally, we have developed a new electron microscopic method in collaboration with Richard Leapman using darkfield STEM tomography for sections up to 300-400 nm thick to provide detailed reconstructions of whole PSDs. The darkfield imaging, which provides enhanced visualization of weakly stained small structures at the PSDs, provides an opportunity to reconstruct detailed molecular organization of more or less complete PSDs in intact neurons. A new initiative is an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility. Dr. Smith has cultured a primitive animal, Trichoplax, that is remarkable in that it lacks synapses, but shows behavior indicative of neural function. These results appear to signify an early stage in evolving nervous systems, prior to the development of synapses, that utilizes peptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. A cell that senses a direction of gravity and mediates behavior accordingly has also been discovered, but what control systems are utilized is not yet clear. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors is expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains.

兴奋性谷氨酸突触的突触后密度（PSD）是一个巨大的分子机器，是记忆、信息处理和存储的关键部位。为了探索PSD的详细分子组织，我们开发了一种方法来冷冻替代海马培养物，然后通过EM断层扫描在薄片上检查它们，以显示PSD内接近其自然设置的单个蛋白质复合物。利用断层扫描的初步工作显示，PSD的核心是一组膜相关的垂直定向细丝，其中含有支架蛋白PSD-95，呈扩展结构和极化方向，其n端位于突触后膜。这一发现提供了对PSD核心结构的整体组织的深入了解，因为支架蛋白，如PSD-95家族MAGUK蛋白，具有不同的多个不同的结合位点，可以沿其长度排列其他蛋白质。因此，PSD-95的规则排列对包括谷氨酸受体在内的许多其他PSD蛋白施加了排序，并为PSD的结构提供了总体规划。