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.

项目总结/摘要 内在无序蛋白（IDP）是细胞内自组装的驱动因素。由高度多价的 通过相互作用，IDP组织由液-液相控制的亚细胞组装体（生物分子凝聚物） 分离动力学（LLPS）。从基因组组织到突触可塑性，生物分子凝聚物 影响广泛的细胞机制。尽管这些令人兴奋的见解，生物物理和生理 对底层IDP组件的性质仍然知之甚少。这种知识差距是普遍存在的 因为研究IDP及其LLPS的现有工具需要非生理条件。的主要挑战 是国内流离失所者对环境的敏感性。他们的LLPS行为是不可预测的改变， 环境和生物化学变化，包括翻译后修饰（PTM）和分子 用荧光蛋白标记。需要新的工具来解剖生物分子凝聚物在其自然状态下， 细胞环境，组织内。在IDP组件的组织非破坏性探测方面的进展将 缩小分离IDP生物物理学和IDP相关疾病机制的差距。至关重要的是， 是无法治愈的退行性大脑疾病的病理标志，数十年的历史和LLPS精炼 观察未能提供机械的见解。在这些挑战的推动下，本提案 推进生物分子传感器探测和操纵细胞内IDP-类脑组织中的组装。的 关键的创新是将超弱和LLPS特异性多价相互作用编码为工程化的 配备有荧光和催化结构域的IDP。由此产生的国内流离失所者将作为多功能LLPS- 传感器，使战略偏离分子标记的本地国内流离失所者。这个工程平台构建了 荧光LLPS传感器最近率先照亮LLPS在皮肤中的动态。通过催化 生物素化和蛋白质解聚，下一代LLPS传感器将使生物分子解剖， IDP组装，并提供用于对抗神经病理性IDP组装的工具。前进和部署 这些创新，该提案将工程师和询问多功能LLPS传感器在国家的最先进的 阿尔茨海默病、额颞叶痴呆和肌萎缩侧索硬化的脑类器官模型。 结合传感器激活的活细胞成像和邻近蛋白质组学，所提出的实验方法 将解决长期存在的关键问题，将病理性IDP组装和主要人类 神经退行性疾病通过在大脑神经病理学建模中加入分子工具和严谨性， 类器官，这项工作将使和刺激分子水平的解剖年龄依赖的人类 神经变性除了产生对IDP驱动的神经变性机制的治疗见解， 这一建议将推动一个广泛适用的传感器-类器官平台，以研究生物分子凝聚物 跨越生物系统。