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

项目摘要 这个R21的目标是开发新的工具，用于揭示力启动的基因转录激活机制。 在核机械转导过程中，使用组合的单细胞力学和位点特异性荧光， cence报告。核机械力转导是指外力通过细胞核直接激活基因。 骨骼力传递到染色质。这一过程对发展和 维持承重结构组织，如软骨、肌腱、韧带和相关的结缔组织， 分泌组织异常的机械传导有助于骨关节炎的病理生理学。显著 我们对核机械传导的理解存在空白，尤其是在真实的分子水平上 时间最重要的事件是机械力转化为转录的关键时刻- 在染色质锚定到核内膜的连接处基因的正常激活。 这一事件的知识将大大促进我们对核机械转导的理解， 建立机械线索与下游基因激活的系统相关性。这将使 在通过机械线索编程细胞表型方面的影响和新应用，以及新的 theragnostic工具。 许多机械反应基因在短时间内迅速激活，通常是几秒钟或几秒钟。 分钟，表明它们在转录上暂停。它被确定为转录的快速激活- 暂停的基因需要转录因子细胞周期蛋白依赖性激酶9（Cdk 9）。最近，Cdk 9 被定位在内核膜上。我们的中心假设是Cdk 9的激酶活性可以 可用作在原位真实的时间成像机械响应基因激活的替代物（图1）。 在这里，我们建议开发新的荧光报告监测Cdk 9的活动，定位在内核 膜，使用单细胞力学激活后。 我们的新工具将使我们能够可视化基因激活原位，在活细胞中通过我们的真实的时间。 位点特异性荧光报告分子在单细胞水平上的机械扰动。两个具体目标是： (SA1)开发并验证Cdk 9活性报告基因作为成像基因激活的替代物。(SA2)Corre- 利用Cdk 9报告基因在活细胞中的基因激活的晚期机械提示参数，使用单细胞 力学和改变施加力的位置、大小、持续时间、方向和频率。 概念上的创新是，Cdk 9活动的报告者充当了可视化力初始的替代者， 基因激活。技术创新包括设计和生产特定地点的Cdk 9荧光剂- cence报告者，使用单细胞机制来传递设计的机械提示的组合方法， 和AFM/共聚焦成像，以监测随后的基因激活在真实的时间和原位。 结果包括：（a）可视化活细胞中的真实的时间和原位核机械转导; (b)捕获基因的转录激活;（c）揭示信号网络，包括核骨架的接头， 细胞核和细胞骨架（LINC）复合物、核被膜蛋白和核纤层蛋白，它们将染色质锚在细胞核上。 核周基质;和（d）建立转录激活与机械线索的相关性，镁，镁 强度、持续时间、力的方向（剪切力与法向力）、力调制频率和振幅。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， 化学线索，并产生一个新的和有影响力的工具，为科学界探索细胞前体， 参与核机械转导的细胞。

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.