On-demand neuronal condensate interactomes using new optogenomics tools

项目总结 生物分子冷凝物(Bmc)正在成为一种无处不在的区隔特征。 在细胞内。这些结构可以通过特定蛋白质的液-液相分离和 从细胞质或核质的其余部分中分离出来的RNA，可以发挥重要的功能(如 酶反应，信号调节)。RNA结合蛋白TDP-43的骨髓细胞是 肌萎缩侧索硬化症的假说是使病理性聚集体成核并导致神经元死亡 侧索硬化(ALS)。然而，用传统技术很难描述骨髓基质细胞。 因为它们没有膜，可以在稀释后溶解，因此具有挑战性(或 不可能)提纯。此外，询问与疾病相关的BMCs的毒性效应 由于难以控制类液体或类固体的诱导而产生问题 在神经元内凝结。在这里，我们建议开发一个新的技术平台， 阶段-序列，它结合了来自不同领域的两种方法-生物物理-细胞内 蛋白质缩合的重建和RNA组织的基因组图谱--以解决 目前研究神经元中骨髓细胞的方法限制。这款工具结合了光生技术 用尖端基因组学方法(SPRITE)诱导神经元(核小体)中的骨髓细胞 询问骨髓细胞的RNA组成和功能。首先，我们将为以下工作奠定基础 通过在初级神经元中建立Sprite来创建RNA-Sequence的全基因组图谱 RNA在这些高度专门化的细胞中的相互作用，并确认我们可以在 通过诱导应激颗粒诱导单个骨髓细胞。然后我们将开发早期和后期的模型 通过在有核细胞的神经元中诱导TDP-43骨髓细胞来实现ALS病理，这是一项新技术，可以 用于控制凝析油的光生诱导，具有严格的时间和空间分辨率。 最后，我们将结合Sprite和神经元中的Colet来创建一个新的平台--PhaseSeq 用于同时诱导BMC并通过作图RNA鉴定其功能 选民。这些实验将使我们能够获得关于RNA组织的宝贵见解 神经元BMCs，确定TDP-43凝聚体如何导致神经元死亡，并识别 肌萎缩侧索硬化症治疗新途径。我们预计，阶段顺序平台将广泛 适用于探讨神经元内源性BMCs的功能及其后果 疾病中异常的骨髓细胞。

PROJECT SUMMARY Biomolecular condensates (BMCs) are emerging as a ubiquitous feature of compartmentalization within cells. These structures can form by liquid-liquid phase separation of particular proteins and RNAs from the rest of the cytoplasm or nucleoplasm and can perform important functions (e.g. enzymatic reactions, signaling regulation). BMCs of the RNA-binding protein TDP-43 are hypothesized to nucleate pathological aggregates and lead to neuron death in Amyotrophic Lateral Sclerosis (ALS). However, BMCs are difficult to characterize with traditional techniques because they lack membranes and can dissolve upon dilution and, therefore, are challenging (or impossible) to purify. Additionally, interrogating toxic effects of disease-associated BMCs has been problematic because of the difficulty of controlled induction of liquid-like or solid-like condensates within neurons. Here, we propose the development of a new technology platform, Phase-seq, that combines two approaches from different fields – biophysical in-cellulo reconstitution of protein condensation and genomic mapping of RNA organization – to address current methodological limitations for studying BMCs in neurons. This tool combines optogenetic induction of BMCs in neurons (Corelets) with a cutting-edge genomics approach (SPRITE) to interrogate the RNA compositions and functions of BMCs. First, we will lay the groundwork for Phase-seq by establishing SPRITE in primary neurons to create a genome-wide map of RNA- RNA interactions in these highly specialized cells and confirm that we can identify RNAs within individual BMCs by inducing stress granules. We will then develop models of early and late stage ALS pathology by inducing TDP-43 BMCs in neurons with Corelets, a new technology that allows for controlled optogenetic induction of condensates with tight temporal and spatial resolution. Finally, we will combine SPRITE and Corelets in neurons to create Phase-seq, a novel platform for simultaneous induction of BMCs and characterization of their functions by mapping RNA constituents. These experiments will allow us to gain valuable insight into RNA organization in neuronal BMCs, determine how TDP-43 condensates may lead to neuron death, and identify novel therapeutic avenues for ALS. We anticipate that the Phase-seq platform will be broadly applicable for interrogating the functions of endogenous BMCs in neurons and the consequences of aberrant BMCs in disease.