EuroSYNAPSE - Spatio-temporal organization of the synaptic membrane for synaptic vesicle protein recycling

EuroSYNAPSE - 用于突触小泡蛋白质回收的突触膜时空组织

• 批准号: 128368325
• 负责人: Professor Volker Haucke, Ph.D.
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/128368325?language=en
• 关键词: EuroSYNAPSE; Spatio; temporal; organization; synaptic

The presynaptic terminal contains clusters of synaptic vesicles (SVs), key organelles of chemical neurotransmission (1). Proteomics data indicate that SVs comprise distinct sets of proteins and lipids present in defined stoichiometries (2) which must be maintained during repetitive rounds of exo- and endocytosis. How this precise sorting of SV membrane proteins is accomplished molecularly is not well understood. SV components could remain clustered during their exo-endocytic itinerary as suggested by high-resolution stimulated emission-depletion (STED) microscopy (3), or be reclustered at the cell surface (4) by individual sorting via recognition of sorting determinants by cargo-specific adaptor proteins such as AP-2, stonin2/stoned B (5, 6), AP180 and perhaps others. SV exo- and endocytosis appear to be temporally coupled but spatially segregated between the active zone (sites of fusion) and the surrounding endocytic or periactive zone. The tight temporal coupling between SV exo- and endocytosis (7) suggests that SV cargo protein sorting and recycling likely involves precise control of the localization and dynamics of endocytic proteins or protein complexes as they partition between sites of fusion (active zone) and endocytosis (periactive zone) (8). How the spatio-temporal dynamics of endocytic proteins at the nerve terminal are controlled is largely unknown but likely involves membrane-associated multidomain scaffolding proteins organizing the periactive zone (7, 9). While recent years have witnessed enormous progress in the identification and characterization of the cytomatrix assembled at the active zone (CAZ, also termed presynaptic grid) comparably little is known about the structural components and the functional organization of the periactive zone that surrounds it (7). From a simplified perspective the periactive zone might be viewed as a three-layered structure comprised of a pool of surface stranded SV cargo, dynamic assemblies of endocytic proteins, and largely immobile scaffolds such as the giant protein piccolo. However, the precise underlying mechanisms for this organization have not been unravelled. Movement of endocytic proteins to and from the periactive zone during SV cycling is accompanied by the concomitant assembly of clathrin-coated structures. This process involves the coordinated formation of a regulated network of protein-protein and protein-lipid interactions. The endocytic network appears to be organized around central hubs, i.e. factors that at a given time or within a defined space display a disproportionately high number of interactions (10). As maturation of the endocytic vesicle progresses, the network proceeds from a SV cargo and phosphatidylinositol (4,5)-bisphosphate (PIP2)- to an AP-2-, and finally to a clathrin-centered state (6, 10). A number of studies indicate that this progression involves the regulated relocalization of endocytic proteins or complexes thereof to sites of SV endocytosis (1, 8). Ultrastructural and light microscopy data in combination with dominant-negative approaches have revealed that molecularly similar components such as the SH3 domain containing proteins amphiphysin, syndapin, endophilin, and intersectin are recruited to the periactive zone and play non-overlapping functional roles in SV recycling. Based on combined genetic, biochemical, and functional studies endocytic proteins might be grouped into functional protein modules, i.e. macromolecular complexes defined by their physical interactions within a characteristic time domain and by their spatial sequestration (11). For example, the scaffolding protein intersectin physically and functionally interacts with eps15, dynamin, and synaptojanin. Mutants of eps15 and intersectin in D. melanogaster phenotypically resemble each other (12) and both proteins co-migrate from the centre of synaptic boutons to the periactive zone in response to activity, suggesting that intersectin together with eps15 is part of a functional module that regulates localization and activity of dynamin and synaptojanin at late stages of clathrin-mediated SV endocytosis (Figure 1B). At present we do not understand how such movement is accomplished at the molecular level but interactions with the actin cytoskeleton are likely to be involved. As endocytic proteins can be associated with several modules intersectin and eps15 via their EH domains are also part of a functional module containing the synaptotagmin-specific sorting adaptor stonedB/ stonin2, its interactor GIT1 (our own unpublished data), AP180, and the AP-2 complex implicated in retrieval of surface-stranded pools of SV cargo from the presynaptic plasmalemma (Figure 1A). Thus, functional endocytic protein modules may be viewed as dynamic components of the periactive zone that are under tight spatio-temporal control. How such regulatory control is accomplished and how it is synchronized with the migration of the vesicle membrane and associated components after fusion remains unknown (9), largely because current methodology allows either subcellular imaging without access to the time domain (i.e. by conventional electron microscopy) or fast live cell imaging based on light microscopic techniques that do not provide sufficient spatial resolution. The work described in this proposal aims to tackle these problems and will thus enable us to gain unprecedented insights into the spatio-temporal organization of the synaptic membrane for clathrin-mediated SV protein recycling.

突触前末梢含有突触囊泡（SV）簇，这是化学神经传递的关键细胞器（1）。蛋白质组学数据表明，SV包含以确定的化学计量存在的不同的蛋白质和脂质组（2），其必须在重复的胞吞和胞吞循环期间维持。SV膜蛋白的这种精确分选是如何在分子水平上完成的还不清楚。SV组分可在其外吞-内吞行程期间保持成簇，如高分辨率受激发射耗尽（STED）显微镜所示（3），或通过货物特异性衔接蛋白（如AP-2、stonin 2/stoned B（5，6）、AP 180等）识别分选决定簇而在细胞表面重新成簇（4）。SV外吞和内吞似乎是时间上耦合的，但在空间上分离的活性区（融合位点）和周围的内吞或周活性区之间。SV外吞和内吞之间的紧密时间耦合（7）表明SV货物蛋白分选和再循环可能涉及精确控制内吞蛋白或蛋白复合物的定位和动力学，因为它们在融合位点（活性区）和内吞位点（周活性区）之间分配（8）。神经末梢的内吞蛋白的时空动力学如何被控制在很大程度上是未知的，但可能涉及组织活动周区的膜相关多结构域支架蛋白（7，9）。虽然近年来在活动区（CAZ，也称为突触前网格）组装的细胞基质的识别和表征方面取得了巨大进展，但对围绕它的活动周区的结构成分和功能组织知之甚少（7）。从简化的角度来看，周活性区可以被看作是一个三层结构，包括一个表面链SV货物池，内吞蛋白的动态组装，以及很大程度上不动的支架，如巨大的蛋白短笛。然而，该组织的确切基本机制尚未解开。在SV循环过程中，内吞蛋白质往返于外周活动区的运动伴随着网格蛋白包被结构的伴随组装。该过程涉及蛋白质-蛋白质和蛋白质-脂质相互作用的调节网络的协调形成。内吞网络似乎是围绕中心枢纽组织的，即在给定时间或在限定空间内显示不成比例的高数量相互作用的因子（10）。随着内吞囊泡的成熟进行，网络从SV货物和磷脂酰肌醇（4，5）-二磷酸（PIP 2）-进行到AP-2-，并且最终进行到网格蛋白中心状态（6，10）。许多研究表明，这种进展涉及内吞蛋白或其复合物到SV内吞作用位点的调节性再定位（1，8）。超微结构和光学显微镜的数据与显性负性的方法相结合，揭示了分子相似的成分，如SH 3域含有蛋白质amphiphysin，syndapin，endophilin，和intersectin被招募到围活动区，并发挥非重叠的SV回收功能的作用。基于遗传、生物化学和功能的综合研究，内吞蛋白可能被分组为功能蛋白模块，即由其在特征时域内的物理相互作用和空间封存定义的大分子复合物（11）。例如，支架蛋白intersectin在物理和功能上与eps 15、发动蛋白和synaptojanin相互作用。突变体eps 15和intersectin在D.在表型上，黑腹动物彼此相似（12），并且两种蛋白质响应于活性从突触结的中心共迁移到活动周区，这表明intersectin与eps 15一起是在网格蛋白介导的SV内吞作用的晚期调节发动蛋白和突触Janin的定位和活性的功能模块的一部分（图1B）。目前，我们还不清楚这种运动是如何在分子水平上完成的，但可能涉及与肌动蛋白细胞骨架的相互作用。由于内吞蛋白可以通过它们的EH结构域与几个模块相关联，所以intersectin和eps 15也是包含突触结合蛋白特异性分选接头stonedB/stonin 2、其相互作用物GIT 1（我们自己未发表的数据）、AP 180和AP-2复合物的功能模块的一部分，AP-2复合物涉及从突触前质膜取回SV货物的表面链池（图1A）。因此，功能性内吞蛋白模块可以被视为处于严格时空控制下的活性区的动态组分。这种调节控制是如何实现的以及它如何与融合后囊泡膜和相关组分的迁移同步仍然是未知的（9），这主要是因为目前的方法允许亚细胞成像而不进入时域（即通过常规电子显微镜）或基于光显微镜技术的快速活细胞成像，所述光显微镜技术不提供足够的空间分辨率。本提案中描述的工作旨在解决这些问题，从而使我们能够对网格蛋白介导的SV蛋白再循环的突触膜的时空组织获得前所未有的见解。