Multifunctional phase sensors for probing and manipulation of intracellular biomolecular condensates

用于探测和操纵细胞内生物分子凝聚物的多功能相位传感器

• 批准号: 10473107
• 负责人: Felipe Garcia Quiroz
• 金额: $ 140.85万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473107
• 关键词: Address; Alzheimer' s disease model; Amyotrophic Lateral Sclerosis; Behavior; Biochemical; Biophysics; Biotinylation; Brain; Brain Diseases; Catalytic Domain; Disease; Dissection; Engineering; Environment; Frontotemporal Dementia; Genomics; Human; Knowledge; Link; Liquid substance; Modeling; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Organoids; Pathologic; Phase; Physiological; Post-Translational Protein Processing; Property; Proteins; Proteomics; Skin; Synaptic plasticity; Technology; Therapeutic; Tissues; Work; age related; biological systems; innovation; insight; link protein; live cell imaging; neuropathology; next generation; prevent; self assembly; sensor; tool

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.

项目摘要/摘要 固有的蛋白质（IDP）是细胞内自组装的驱动因素。由高度多价动力 IDP相互作用，组织了亚细胞组件（生物分子冷凝水），该组件受液态液相控制的 分离（LLP）动力学。从基因组组织到突触可塑性，生物分子冷凝水 影响广泛的细胞机制。尽管有这些令人兴奋的见解，但生物物理和生理学 基础IDP组件的特性仍然很少理解。这个知识差距无处不在 因为现有的研究IDP及其LLP的工具需要非生理条件。主要挑战 是IDP的明显环境灵敏度。他们的LLP行为被不可预测 环境和生化变化，包括翻译后修饰（PTM）和分子 用荧光蛋白标记。需要新工具来剖析其本地的生物分子冷凝物 细胞环境，组织内。在组织非干扰性探测IDP组件中的进展将 缩小分隔IDP生物物理学和IDP连接疾病机制的差距。至关重要的是IDP组件 是不可治疗的退化性脑疾病的病理标志，数十年且有LLP的脑部疾病 观察结果未能提供机械见解。受这些挑战的动机，该提议 进步生物分子传感器，以探测和操纵细胞内IDP分配在脑样组织中。这 关键创新是对工程的超湿和LLPS特异性多价交互的编码 配备荧光和催化域的IDP。最终的IDP将用作多功能LLP- 传感器，使人可以与本机IDP的分子标记进行战略偏离。这个工程平台构建 在荧光LLPS传感器上，最近率先启用了皮肤中的LLPS动力学。通过催化 生物素化和蛋白质 - 分散，下一代LLPS传感器将使生物分子解剖 IDP组件并提供用于对抗神经病理IDP组件的工具。进步和部署 这些创新，该提案将在最新技术中设计和询问多功能LLPS传感器 阿尔茨海默氏病，额颞痴呆和肌萎缩性侧索硬化症的脑器官模型。 结合了启用传感器的活细胞成像和接近蛋白质组学，提出的实验方法 将解决将病理IDP组件和主要人类的重要问题解决的长期关键问题 神经退行性疾病。通过将分子工具和严格添加到大脑中神经病理学的建模中 类器官，这项工作将使并刺激依赖年龄的人的分子级解剖 神经变性。除了对IDP驱动的神经变性机制产生治疗见解， 该提案将推进一个广泛适用的传感器 - 甲然平台，用于研究生物分子冷凝水 跨生物系统。