Multifunctional phase sensors for probing and manipulation of intracellular biomolecular condensates

项目摘要/摘要 固有无序蛋白(IDPs)是细胞内自组装的驱动力。由高度多价提供动力 相互作用，IDPs组织由液-液相控制的亚细胞组件(生物分子凝聚体) 分离(LLP)动力学。从基因组组织到突触可塑性，生物分子凝聚 影响广泛的细胞机制。尽管有这些令人兴奋的见解，但生物物理学和生理学 基本的IdP-组装的属性仍然知之甚少。这种知识鸿沟无处不在。 因为现有的研究国内流离失所者及其有限合伙人的工具需要非生理条件。面临的主要挑战 是境内流离失所者对环境的明显敏感性。他们的有限责任合伙人行为被不可预测地改变了 环境和生化变化，包括翻译后修饰(PTM)和分子 用荧光蛋白进行标记。需要新的工具来剖析天然生物分子凝析油 细胞环境，在组织内。在组织内无干扰探测IDP-组件的进展将 弥合分离IdP生物物理学和IdP相关疾病机制的差距。至关重要的是，虽然Idp-组装 是无法治愈的退行性大脑疾病的病理特征，数十年前和精炼的LLP 观察未能提供机械性的见解。在这些挑战的推动下，这项提案 先进的生物分子传感器来探测和操纵类脑组织中的细胞内IDP组装。这个 关键的创新是将超弱和特定于LLP的多价相互作用编码为工程 国内流离失所者配备了荧光区和催化区。由此产生的国内流离失所者将作为多功能有限责任合伙人- 传感器，实现了对本地国内流离失所者的分子标记的战略性偏离。这个工程平台建立了 关于荧光LLP-传感器最近率先用于照亮LLP在皮肤中的动态。通过催化 生物素化和蛋白质解聚，下一代LLP传感器将使生物分子解剖成为可能 Idp-集合体，并提供对抗神经病理性Idp集合体的工具。推进和部署 这些创新，这项计划将设计和讯问多功能LLP-传感器在最先进的状态 阿尔茨海默病、额颞叶痴呆和肌萎缩侧索硬化症的脑器官模型。 结合传感器启用的活细胞成像和邻近蛋白质组学，提出的实验方法 将解决长期存在的关键问题，将病理性的IDP-组装与主要的人类 神经退行性疾病。通过将分子工具和严格性添加到脑内神经病理学的建模中 有机化合物，这项工作将使和刺激分子水平的年龄依赖的人类解剖 神经退行性变。除了对IDP驱动的神经退行性变机制产生治疗见解外， 这一提议将推动一种广泛适用的传感器-有机平台来研究生物分子冷凝物 跨越生物系统。

Project Summary/Abstract Intrinsically-disordered proteins (IDPs) are drivers of intracellular self-assembly. Powered by highly multivalent interactions, IDPs organize subcellular assemblies (biomolecular condensates) governed by liquid-liquid phase separation (LLPS) dynamics. From genomic organization to synaptic plasticity, biomolecular condensates influence wide-ranging cellular mechanisms. Despite these exciting insights, the biophysical and physiological properties of the underlying IDP-assemblies remain poorly understood. This knowledge gap is pervasive because existing tools to study IDPs and their LLPS require non-physiological conditions. The major challenge is the pronounced environmental sensitivity of IDPs. Their LLPS behavior is unpredictably altered by environmental and biochemical changes, including post-translational modifications (PTMs) and molecular tagging with fluorescent proteins. New tools are needed to dissect biomolecular condensates in their native cellular environments, within tissues. Progress towards in tissue non-disruptive probing of IDP-assemblies will close the gap separating IDP biophysics and IDP-linked disease mechanisms. Crucially, while IDP-assemblies are pathological hallmarks of untreatable degenerative brain disorders, decades-old and LLPS-refined observations have failed to provide mechanistic insights. Motivated by these challenges, this proposal advances biomolecular sensors to probe and manipulate intracellular IDP-assemblies in brain-like tissues. The crucial innovation is the encoding of ultra-weak and LLPS-specific multivalent interactions into engineered IDPs equipped with fluorescent and catalytic domains. The resulting IDPs will serve as multifunctional LLPS- sensors, enabling a strategic departure from molecular tagging of native IDPs. This engineering platform builds on fluorescent LLPS-sensors recently pioneered to illuminate LLPS dynamics in skin. By catalyzing biotinylation and protein-disaggregation, next-generation LLPS-sensors will enable biomolecular dissection of IDP-assemblies and provide tools for combating neuropathological IDP-assemblies. To advance and deploy these innovations, this proposal will engineer and interrogate multifunctional LLPS-sensors in state-of-the-art brain organoid models of Alzheimer's disease, frontotemporal dementia, and amyotrophic lateral sclerosis. Combining sensor-enabled live cell imaging and proximity proteomics, the proposed experimental approaches will address long-standing key questions linking pathological IDP-assemblies and major human neurodegenerative disorders. By adding molecular tools and rigor to the modeling of neuropathology in brain organoids, this work will enable and stimulate molecular-level dissection of age-dependent human neurodegeneration. Beyond generating therapeutic insights into IDP-driven mechanisms of neurodegeneration, this proposal will advance a broadly applicable sensor-organoid platform to study biomolecular condensates across biological systems.