Single molecule biomolecular condensate analysis in neurons

项目摘要/摘要 生物分子冷凝物(BMC)定义了分离特定基因的无膜小细胞隔间。 蛋白质和核酸来产生细胞功能。光漂白后荧光恢复(FRAP)是 目前，选择的方法是在体外和体内表征骨髓基质细胞，并得出平均扩散和 捕获捕获在这些冷凝物中的分子。尽管FRAP是检索这些重要信息的好方法 它极大地限制了我们对活细胞和细胞中骨髓细胞纳米尺度组织和动力学的理解 排除了对纳米级BMCs(低于200 nm的光的衍射)的分析。单分子成像 超分辨率显微镜可以直接成像捕获在BMC中的分子，具有更大的 在体外和在活神经元中的时空分辨率，并有可能揭示纳米级的骨髓细胞和 它们在老龄化和阿尔茨海默病(AD)背景下的演变。然而，目前还没有 生物信息学工具，在单分子水平上专门分析骨髓细胞。我们开发了一种新的工具， 命名为纳米级时空索引集群(NACTIC)(Wallis等人，BioRxiv，2021，参考文献7), 为蛋白质在液-液相过程中的动力学提供了前所未有的洞察力 在大型和纳米尺度的骨髓细胞中的分离/过渡，在活的神经元中。我们鉴定了牛磺酸和突触核蛋白 他们在海马神经元中产生突触骨髓细胞的能力。纳斯特拉分析显示， Tau分子确实可以在活的海马神经元中形成小的纳米簇，在那里它们表现得非常低。 流动性和对1，6-己二醇的敏感性，1，6-己二醇是一种脂肪醇，用于抑制弱分子相互作用 调解骨髓细胞。纳斯泰克将是我们能力的核心，为理解做出重大贡献 因为它提供了前所未有的机会来研究细胞的时空行为 这些冷凝物中的分子。NACTIC将进一步发展到包括双色单分子 分析。这将有助于评估这些纳米尺度的骨髓基质细胞与它们的 2和3D中的蜂窝环境。我们将使用成对的对偶来验证我们的双色Nastic实现 Tau野生型和AD突变体P301L和α-突触核蛋白的成像。这将使我们能够破译共同聚集 动力学并检查一个纳米BMC对另一个的潜在层次依赖关系，从而实现精细化 了解突触中纳米级骨髓细胞的产生。我们预计P301L突变将 显著增加Tau纳米BMCs的大小和寿命，并改变它们与其他突触的关系 分子。 这笔赠款的结果将是开发和验证一条分析管道，以使 活体海马神经元中Tau和α-突触核蛋白纳米BMCs时空关系的探讨。 因此，我们的项目将产生数据和急需的技术，为我们的 了解骨髓细胞在神经元功能和阿尔茨海默病中的作用。

PROJECT SUMMARY/ABSTRACT Biomolecular condensates (BMCs) define small membraneless cellular compartments that segregate specific proteins and nucleic acids to generate cellular functions. Fluorescence recovery after photobleaching (FRAP) is currently the method of choice to characterize BMCs in vitro and in vivo and derive the average diffusion and trapping of molecules trapped in these condensates. Although FRAP is a good way to retrieve these important metrics, it greatly limits our understanding of BMCs nanoscale organization and dynamics in live cells and precludes the analysis of nanoscale BMCs (below the diffraction of light ~200 nm). Single-molecule imaging super-resolution microscopy allows direct imaging of molecules trapped in BMCs with much greater spatiotemporal resolution both in vitro and in live neurons, and has the potential to reveal nanoscale BMCs and their evolution in the context of ageing and Alzheimer’s disease (AD). However, there are currently no bioinformatic tools to specifically analyze BMCs at the single-molecule level. We have developed a novel tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), (Wallis et al., bioRxiv, 2021, ref. 7), which offers unprecedented insights into the dynamics of proteins undergoing liquid-liquid phase separation/transition in large and nanoscale BMCs, in live neurons. We identified Tau and synuclein as candidates for their ability to generate synaptic BMCs in hippocampal neurons. NASTIC analysis reveals that Tau molecules can indeed form small nanoclusters in live hippocampal neurons in which they exhibit very low mobility and sensitivity to 1,6-hexanediol, an aliphatic alcohol used to inhibit weak molecular interactions that mediate BMCs. NASTIC will be at the core of our ability to make a significant contribution to the understanding of BMCs in neurons because it offers unprecedented opportunities to examine the spatiotemporal behaviour of molecules in these condensates. NASTIC will be developed further to encompass two-color single-molecule analysis. This will be instrumental to assess the spatiotemporal relationship of these nanoscale BMCs with their cellular environment in 2 and 3D. We will validate our two-color NASTIC implementation with the pairwise dual imaging of Tau wildtype and AD mutant P301L, and a-synuclein. This will allow us to decipher the co-clustering dynamics and examine potential hierarchical dependency of one nanoBMC upon the other allowing refined understanding of the generation of nanoscale BMCs in synapses. We anticipate that the P301L mutation will largely increase the size and lifetime of the Tau nanoBMCs and alter their relationship with other synaptic molecules. The outcome of this grant will be the development and validation of an analytical pipeline that will enable exploration of the spatiotemporal relationship of Tau and a-synuclein nanoBMCs in live hippocampal neurons. Our project will therefore generate data and a much-needed technology opening new avenues for our understanding of BMCs in neuronal function and AD.