Supplement request for Cellular mechanotransduction - from the immune response to transcriptional regulation

由黏附和信号受体介导的细胞-细胞和细胞-底物相互作用是高度动态的和 受制于在界面上施加大量机械力的细胞骨架运动。单元格的组合方式 机械和生化信号执行的具体功能还不是很清楚。受此支持 Mira奖，我们正在研究机械信号如何传递到细胞核来调节基因表达 以功能适当的方式，以及机械线索如何与特定组织的线索相互作用。这需要 一种成像系统，能够在深部以高信噪比进行多色单分子成像 细胞核。高倾斜叠层光学薄片(HILO)显微镜是一种成像技术，它允许 用于以纳米级分辨率显示细胞内部深处的单分子。单人 转录因子迁移率和结合的分子成像提供了基因调控的动态视图 单细胞水平，从而补充高通量基因组研究。在初步研究中，我们已经表明 转录因子和组蛋白表现出多种低迁移率状态，表明存在复杂的相互作用 染色质的异质动力学和Tf与这些染色质移动模式的结合之间。按顺序 为了研究细胞如何整合机械和化学信号，我们正在使用硬度可调的水凝胶来 研究机械线索对转录的调节。而传统的全内反射 具有荧光(TIRF)能力的显微镜可以适用于玻璃盖片上的细胞的HELO成像， 水凝胶(30-50微米厚度)上的成像细胞由于厚度和过剩带来了挑战 光在中间凝胶中的散射。此外，在我们的设置限制内的相对低功率激光器 在快速成像速率下跟踪单分子所需的信噪比，使分析 亚秒时间尺度下的TF扩散和染色质动力学是困难的。此补充请求是为了升级 通过增加一个先进的方位TIRF来增强我们目前的TIRF显微镜的高光显微镜能力 模块、更高功率的激光器和像素寄存的多色单分子成像模块。作为单身人士 分子成像与我们最初的目标直接相关，这种增强的能力将告诉我们所有的 建议进行的研究。此外，高速成像能力将使我们能够应用我们最新开发的 研究转录因子迁移和染色质相互作用的算法。我们对机械传感的研究 以及通过细胞微环境的机械特性和细胞内的 潜在的途径将促进我们对乳腺癌和其他癌症的理解。

Cell-cell and cell-substrate interactions, mediated by adhesion and signaling receptors, are highly dynamic and subject to cytoskeletal movements that impart substantial mechanical force at the interface. How cells combine mechanical and biochemical signals to carry out specific functions is not well understood. Supported by this MIRA award, we are examining how mechanical cues are relayed to the nucleus to regulate gene expression in a functionally appropriate manner and how mechanical cues interact with tissue-specific cues. This requires an imaging system capable of multicolor single-molecule imaging with high signal to noise ratio deep within the cell nucleus. Highly Inclined Laminated Optical Sheet (HILO) Microscopy is an imaging technology that allows for visualization of single molecules deep within the cell interior with nanometer scale resolution. Single molecule imaging of transcription factor mobility and binding yields a dynamic view of gene regulation at the single cell level, thus complementing high throughput genomic studies. In preliminary studies, we have shown that transcription factors and histones exhibit multiple low mobility states, indicative of a complex interaction between the heterogeneous dynamics of chromatin and TF binding to these chromatin mobility modes. In order to study how cells integrate mechanical and chemical cues, we are using hydrogels of tunable stiffness to examine the regulation of transcription by mechanical cues. While conventional Total Internal Reflection Fluorescence (TIRF) capable microscopes can be adapted to HILO imaging of cells on glass coverslips, imaging cells on hydrogels (30-50 micron thickness) poses a challenge due to the thickness and the excess scattering of light within the intervening gel. Furthermore, the relatively low power lasers within our setup limits the signal to noise ratios necessary for tracking single molecules at fast imaging rates, making the analysis of TF diffusion and chromatin dynamics at sub-second timescales difficult. This supplement request is to upgrade the HILO microscopy capabilities of our current TIRF microscope by adding an advanced azimuthal TIRF module, higher power lasers and pixel-registered multicolor single molecule imaging modules. As single molecule imaging is directly related to all of our original Aims, this enhanced capability will inform all our proposed studies. Moreover, the high speed imaging capability will enable us to apply our newly developed algorithms to study transcription factor mobility and chromatin interactions. Our studies of mechanosensing and the regulation of transcription by mechanical properties of the cellular microenvironment and the underlying pathways will advance our understanding of breast and other cancers.