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.

项目概要/摘要 本质无序蛋白 (IDP) 是细胞内自组装的驱动因素。由高度多价提供动力 相互作用，IDP 组织受液-液相控制的亚细胞组装体（生物分子凝聚体） 分离（LLPS）动力学。从基因组组织到突触可塑性、生物分子凝聚体 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 底层 IDP 组件的特性仍然知之甚少。这种知识差距是普遍存在的 因为研究国内流离失所者及其 LLPS 的现有工具需要非生理条件。主要挑战 是国内流离失所者明显的环境敏感性。他们的 LLPS 行为被不可预测地改变 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。需要新的工具来剖析生物分子凝聚体的天然状态 组织内的细胞环境。 IDP 组件的组织无破坏性探测方面的进展将 缩小 IDP 生物物理学和 IDP 相关疾病机制之间的差距。至关重要的是，虽然 IDP 集会 是无法治疗的退行性脑部疾病的病理特征，已有数十年历史并经过 LLPS 改进 观察未能提供机制见解。受到这些挑战的推动，本提案 开发生物分子传感器来探测和操纵类脑组织中的细胞内 IDP 组件。这 关键的创新是将超弱和 LLPS 特定的多价相互作用编码到工程中 IDP 配备荧光和催化结构域。由此产生的 IDP 将作为多功能 LLPS- 传感器，从而从战略上摆脱对本地国内流离失所者的分子标记。该工程平台构建 荧光 LLPS 传感器最近率先用于阐明皮肤中的 LLPS 动态。通过催化 生物素化和蛋白质解聚，下一代 LLPS 传感器将实现生物分子解剖 IDP 组件并提供对抗神经病理性 IDP 组件的工具。推进和部署 这些创新，该提案将设计和研究最先进的多功能 LLPS 传感器 阿尔茨海默病、额颞叶痴呆和肌萎缩侧索硬化症的脑类器官模型。 结合传感器支持的活细胞成像和邻近蛋白质组学，提出的实验方法 将解决将病态的国内流离失所者集会与重大人类活动联系起来的长期存在的关键问题 神经退行性疾病。通过在大脑神经病理学建模中添加分子工具和严谨性 类器官，这项工作将实现并刺激年龄依赖性人类的分子水平解剖 神经变性。除了对 IDP 驱动的神经变性机制产生治疗见解之外， 该提案将推进一个广泛适用的传感器类器官平台来研究生物分子凝聚物 跨生物系统。