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

项目总结 该R21的目标是开发新的工具来揭示力启动的基因转录介质的激活。 中国在核力学转导过程中，使用结合单细胞力学和特定部位的荧光- Cence报道。核机械转导被定义为通过外力通过细胞外刺激直接激活基因。 骨骼力量传播到染色质。这一过程对于发展和 维护承重结构组织，如软骨、肌腱、韧带和相关的关节。 不可缺少的组织。异常的机械转导参与了骨关节炎的病理生理学。意义重大 我们对核力学转导的理解存在空白，特别是在真实的分子水平上 时间到了。最重要的事件是机械力转化为转录的关键时刻。 在染色质与内核膜连接的连接处基因的选择性激活。 对这一事件的了解将极大地促进我们对核力学转导和 建立机械信号与下游基因激活的系统关联。这会有很宽的 机械线索对细胞表型编程的影响和新的应用 不可知论工具。 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，这表明它们在转录上暂停了。现已证实，转录-2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(CDK9)。最近，CDK9已经 定位于内核膜。我们的中心假设是，CDK9的激酶活性可以 被用作实时原位成像机械反应基因激活的替代物(图1)。 在这里，我们建议开发新的荧光记者来监测CDK9的活动，定位于内核 膜，在使用单细胞机制激活时。 我们的新工具将使我们能够通过我们的 单细胞水平机械扰动下的定点荧光报告。两个具体目标是： (SA1)开发和验证CDK9活性报告作为成像基因激活的替代物。(Sa2)相应的- 利用CDK9报告基因在活细胞中的晚期机械提示参数，使用单细胞 力学和改变施加的力的位置、大小、持续时间、方向和频率。 概念上的创新是，CDK9活动的记者服务于可视化力量-INITI的替代品- 激活基因。技术创新包括设计和生产特定部位的CDK9荧光- 由此记者获悉，采用单细胞力学相结合的方法来传递设计的力学线索， 和AFM/共聚焦成像，实时和原位监测后续基因的激活。 结果包括：(A)实时和原位显示活细胞中的核机械转导； (B)捕捉基因的转录激活；。(C)揭示信号网络，包括核糖核酸的连接物。 伊顿和细胞骨架(LINC)复合体，核膜蛋白和将染色质锚定到 核周基质；以及(D)建立转录激活与机械提示的相关性，例如，MAG- 力值、持续时间、力的方向(剪切与法线)、力的调制频率和幅度。这些 结果支持我们的长期目标：(A)通过ME计划基因激活和细胞信号传递- 并为科学界提供一种新的、有影响力的工具来探索细胞亲和力 参与核力学转导的赛斯。

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.