In situ and real-time readout of nuclear mechanotransduction via single cell mechanics and site-specific fluorescence reporting

通过单细胞力学和位点特异性荧光报告原位实时读出核力转导

• 批准号: 10745440
• 负责人: Dominik R Haudenschild
• 金额: $ 37.51万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10745440
• 关键词: Affect; Agonist; Antibodies; Atomic Force Microscopy; Biochemical; CDK9 Protein Kinase; Cartilage; Cell physiology; Cells; Chimeric Proteins; Chromatin; Communities; Complex; Connective Tissue; Cytoskeleton; DNA Polymerase II; Degenerative polyarthritis; Development; Event; Fluorescence; Frequencies; Functional disorder; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Image; Immunofluorescence Immunologic; In Situ; In Situ Hybridization; Knowledge; Lamins; Laser Scanning Confocal Microscopy; Legal patent; Ligaments; Location; Maintenance; Mechanics; Molecular; Monitor; Musculoskeletal; Nanotechnology; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamin; Nuclear Matrix; Outcome; Pathology; Phenotype; Phosphorylation; Phosphotransferases; Process; Production; Proteins; RNA; RNA Polymerase II; Reporter; Reporting; Resolution; Signal Transduction; Site; Technology; Tendon structure; Time; Tissues; Transcriptional Activation; Translating; Ultraviolet Rays; Validation; Venus; Visualization; Weight-Bearing state; Work; cell fixing; confocal imaging; design; emerin; env Gene Products; flavopiridol; inhibitor; innovation; insight; mechanical force; mechanical signal; mechanotransduction; novel; novel strategies; precursor cell; programs; response; tool; transcription factor; weight maintenance

PROJECT SUMMARY The goal of this R21 is to develop new tools for unveiling force-initiated activation of the gene transcription ma- chinery during nuclear mechanotransduction, using combined single-cell mechanics and site-specific fluores- cence reporting. Nuclear mechanotransduction is defined as direct gene activation by external force via cyto- skeletal force propagation to the chromatin. This process is critically important for the development and maintenance of weight-bearing structural tissues such as cartilage, tendons, ligaments, and associated con- nective tissues. Aberrant mechanotransduction contributes to the pathophysiology of osteoarthritis. Significant voids exist in our understanding of nuclear mechanotransduction, especially at the molecular level in real time. The most significant event is the critical moment at which mechanical force is translated into transcrip- tional activation of genes at the junction where chromatin is anchored to the inner nuclear membrane. Knowledge of this event would significantly advance our understanding of nuclear mechanotransduction and establish systematic correlation of mechanical cues with downstream gene activation. This would have wide impacts and novel applications in programming cell phenotype via mechanical cues and development of new theragnostic tools. Many mechanoresponsive genes are rapidly activated within a short timeframe, often seconds or minutes, suggesting that they are transcriptionally paused. It is established that rapid activation of transcrip- tionally paused genes requires the transcription factor cyclin-dependent kinase 9 (Cdk9). Recently, Cdk9 has been localized to the inner nuclear membrane. Our central hypothesis is that the kinase activity of Cdk9 can be used as a surrogate for imaging the activation of mechano-responsive genes in real time in-situ (Fig 1). Here we propose to develop new fluorescent reporters to monitor Cdk9 activity, localized at the inner nuclear membrane, upon activation using single-cell mechanics. Our new tools will enable us to visualize gene activation in situ, in real time among living cells via our site-specific fluorescence reporters upon mechanical perturbation at single cell level. Two specific aims are: (SA1) To develop and validate Cdk9 activity reporters as surrogates for imaging gene activation. (SA2) Corre- late mechanical cue parameters with gene activation in living cells utilizing the Cdk9 reporters, using single-cell mechanics and varying the location, magnitude, duration, direction, and frequency of the applied force. The conceptual innovation is that a reporter for Cdk9 activity serves a surrogate for visualizing force-initi- ated gene activation. The technical innovations include the design and production of site-specific Cdk9 fluores- cence reporters, the combined approach of using single-cell mechanics to deliver the designed mechanical cues, and AFM/confocal imaging to monitor the subsequent gene activation in real time and in-situ. The outcomes include: (a) visualizing real time and in situ nuclear mechanotransduction in living cells; (b) capturing the transcriptional activation of genes; (c) revealing signal network, including linker of nucleoskel- eton and cytoskeleton (LINC) complexes, nuclear envelope proteins and lamins that anchor chromatin to the perinuclear matrix; and (d) establishing correlation of transcriptional activation with mechanical cues e.g., mag- nitude, duration, direction (shear versus normal) of force, force modulation frequency and amplitude. These outcomes support our long-term objectives to (a) program gene activation and cellular signaling via me- chanical cues, and to generate a new and impactful tool for the scientific community to probe the cellular pro- cesses involved in nuclear mechanotransduction.

项目概要 R21 的目标是开发新工具来揭示基因转录过程的强制启动激活。 核机械转导过程中的机械作用，使用组合的单细胞力学和位点特异性荧光- 森报告。核力转导被定义为通过细胞-外力直接激活基因 骨骼力传播到染色质。这个过程对于开发和应用至关重要 维持负重结构组织，如软骨、肌腱、韧带和相关构造 结缔组织。异常的机械转导有助于骨关节炎的病理生理学。重要的 我们对核力转导的理解存在空白，尤其是在实际的分子水平上 时间。最重要的事件是机械力转化为转录的关键时刻 染色质锚定于内核膜的连接处基因的激活。 对这一事件的了解将极大地增进我们对核力转导和 建立机械线索与下游基因激活的系统相关性。这将有广泛的 通过机械线索和新开发的细胞表型编程的影响和新颖应用 治疗诊断工具。 许多机械反应基因在短时间内（通常是几秒或几秒）内迅速激活 分钟，表明它们在转录上暂停了。已确定转录的快速激活 暂停基因需要转录因子细胞周期蛋白依赖性激酶 9 (Cdk9)。最近，Cdk9 定位于内核膜。我们的中心假设是 Cdk9 的激酶活性可以 可用作实时原位对机械响应基因激活进行成像的替代物（图 1）。 在这里，我们建议开发新的荧光报告基因来监测位于内核的 Cdk9 活性 使用单细胞力学激活后的膜。 我们的新工具将使我们能够通过我们的技术在活细胞中实时观察基因激活情况 单细胞水平机械扰动时的位点特异性荧光报告基因。两个具体目标是： (SA1) 开发并验证 Cdk9 活性报告基因作为基因激活成像的替代物。 (SA2) 对应 利用 Cdk9 报告基因在活细胞中进行基因激活的后期机械提示参数，使用单细胞 力学并改变所施加力的位置、大小、持续时间、方向和频率。 概念创新在于，Cdk9 活动的报告者充当了可视化强制启动的替代者。 基因激活。技术创新包括设计和生产特定位点的 Cdk9 荧光剂 cence记者，使用单细胞力学来提供设计的机械线索的综合方法， 以及 AFM/共焦成像，实时原位监测随后的基因激活。 结果包括：(a) 活细胞中实时、原位核力转导的可视化； (b) 捕获基因的转录激活； (c)揭示信号网络，包括核骨架的连接子 eton 和细胞骨架 (LINC) 复合物、核膜蛋白和核纤层蛋白将染色质锚定到 核周基质； (d) 建立转录激活与机械信号（例如磁力信号）的相关性 力的强度、持续时间、方向（剪切与法向）、力调制频率和幅度。这些 结果支持我们的长期目标：(a) 通过 me- 编程基因激活和细胞信号传导 机械线索，并为科学界生成一个新的、有影响力的工具来探索细胞亲 涉及核机械转导的过程。