Dynamic Structural Properties of Synapses

突触的动态结构特性

• 批准号: 10018417
• 负责人: Thomas S Reese
• 金额: $ 91.81万
• 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/10018417
• 关键词: Adult; Affinity; Atlases; Binding; Binding Proteins; Biochemical; Brain; Cell Nucleus; Cell membrane; Chromosomes; Complex; Dendrites; Development; Endoplasmic Reticulum; Genes; Genetic Transcription; Goals; Hippocampus (Brain); Human; Immunoelectron Microscopy; Label; Maintenance; Mediator of activation protein; Microtomy; Molecular Machines; Neurons; Nuclear; Poly A; Positioning Attribute; Postsynaptic Membrane; Property; Proteins; Publishing; RNA Polymerase II; Role; Scaffolding Protein; Sequence Analysis; Signal Transduction; Signaling Molecule; Structure; Subcellular Fractions; Synapses; Western Blotting; Yeasts; brain tissue; calmodulin-dependent protein kinase II; density; experimental study; gene product; genetic regulatory protein; insight; mRNA Expression; molecular dynamics; neuronal cell body; postsynaptic; presynaptic density protein 95; protein expression; receptor; stoichiometry; yeast two hybrid system

Past years focus was on two proteins, densin and FAM81A, both found to be highly enriched in affinity-purified PSD-95 complexes. Immuno-gold labeling of cultured hippocampal neurons shows concentration of densin at PSDs, but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was 27 nm, with the majority of label (90 %) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (treatment with high K+ for 2 min), a condition previously shown to promote major re-organization of PSD components, caused only a slight shift in the median distance of densin label, without any significant change in the overall label density at the PSD. We hypothesize that, under excitatory conditions, the relatively stable densin molecules firmly embedded within the PSD would anchor and target activated CaMKII to its substrates, SynGAP and AIDA-1, located at the PSD core. The product of the gene FAM81A, is a protein of unknown function and, so far, no published articles exist that focus on the gene and/or protein. FAM81A mRNA and protein expression are highest in brain tissue (Human Protein Atlas) and our mass spectrometric analysis of isolated PSDs indicated a stoichiometry of one FAM81A to fourteen PSD-95 (Lowenthal et al 2015). Past years experiments showed that FAM81A protein in brain is expressed late in development, with a post-natal gradual increase in levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain by western immunoblotting indicated a distribution of FAM81A similar to PSD-95, with a drastic enrichment in the PSD fraction. Immuno-electron microscopy on adult brain revealed specific immunogold labeling for FAM81A at a subpopulation of PSDs. The label for FAM81A was concentrated at the outer edge of the PSD core, within 30-40 nm from the postsynaptic membrane. On the other hand, an NCBI Conserved Domain search revealed a sequence on FAM81A similar to the SMC_N (N-terminus of Structural Maintenance of Chromosomes) domain, hinting at an interaction of the protein with chromosomes. Also, preliminary results from our commissioned yeast two hybrid study suggested two nuclear binding partners for FAM81A (polyadenylate-binding protein 2 and mediator of RNA polymerase II transcription subunit 10). In conclusion, while biochemical and EM evidence establish FAM81A as a genuine component of the PSD, sequence analysis and Y2H studies, hint at a role for the protein in the nucleus. Further studies will focus on a possible function of FAM81A as a signaling molecule between synapse and nucleus.

过去几年的重点是两种蛋白质，densin和FAM 81 A，两者都被发现在亲和纯化的PSD-95复合物中高度富集。 免疫胶体金标记的培养海马神经元显示致密蛋白浓度在PSD，但也有一些标记在突触外质膜的索马和树突和内质网。 在PSD处，标记物与突触后膜的中值距离为27 nm，其中大多数标记物（90%）被限制在距突触后膜40 nm内，表明致密蛋白主要定位在PSD核心处。 去极化（高K+处理2分钟），一个条件，以前显示，以促进主要重组的PSD组件，只造成轻微的位移中位距离的致密蛋白标签，没有任何显着的变化，在PSD的整体标签密度。 我们假设，在兴奋性条件下，相对稳定的致密蛋白分子牢固地嵌入PSD内，将锚和靶向激活的CaMKII的底物，SynGAP和AIDA-1，位于PSD的核心。 基因FAM 81 A的产物是一种功能未知的蛋白质，到目前为止，还没有发表的文章关注该基因和/或蛋白质。 FAM 81 A mRNA和蛋白质表达在脑组织中最高（Human Protein Atlas），我们对分离的PSD的质谱分析表明1个FAM 81 A与14个PSD-95的化学计量比（Lowenthal et al 2015）。 过去几年的实验表明，大脑中的FAM 81 A蛋白在发育后期表达，出生后水平逐渐增加，与PSD-95的表达平行。 通过蛋白质免疫印迹法比较来自成人脑的亚细胞级分表明FAM 81 A的分布与PSD-95相似，在PSD级分中急剧富集。 成人大脑的免疫电子显微镜显示特定的免疫金标记FAM 81 A在一个亚群的PSD。 FAM 81 A的标记物集中在PSD核心的外边缘，距离突触后膜30-40 nm内。 另一方面，NCBI保守结构域搜索揭示了FAM 81 A上与SMC_N（染色体结构维持的N端）结构域相似的序列，暗示了蛋白质与染色体的相互作用。 此外，我们委托酵母双杂交研究的初步结果表明，FAM 81 A有两个核结合伴侣（多聚腺苷酸结合蛋白2和RNA聚合酶II转录亚基10的介体）。 总之，虽然生物化学和EM证据确定FAM 81 A是PSD的真正组分，但序列分析和Y2 H研究暗示了该蛋白在细胞核中的作用。 进一步的研究将集中在FAM 81 A作为突触和细胞核之间的信号分子的可能功能。