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

由粘附和信号受体介导的细胞-细胞和细胞-基质相互作用是高度动态的， 经受在界面处施加实质性机械力的细胞骨架运动。细胞如何联合收割机 机械和生物化学信号来执行特定的功能还没有得到很好的理解。受此支撑 MIRA奖，我们正在研究如何机械线索传递到细胞核，以调节基因表达 以及机械提示如何与组织特异性提示相互作用。这需要 一种成像系统，其能够以高信噪比在细胞内深处进行单分子成像， 细胞核高倾斜层压光学片（HILO）显微镜是一种成像技术， 用于以纳米级分辨率观察细胞内部深处的单个分子。单个 转录因子迁移和结合的分子成像产生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。初步研究表明， 转录因子和组蛋白表现出多种低迁移率状态，表明它们之间存在复杂的相互作用， 染色质和TF结合这些染色质流动模式的异质动力学之间的关系。为了 为了研究细胞如何整合机械和化学信号，我们正在使用可调刚度的水凝胶， 研究机械信号对转录的调节。虽然传统的全内反射 具有荧光（TIRF）能力的显微镜可以适于玻璃盖玻片上的细胞的HILO成像， 在水凝胶（30-50微米厚度）上成像细胞由于厚度和过量的水凝胶（30-50微米厚度）而提出了挑战 光在中间凝胶内的散射。此外，在我们的设置限制内，相对低功率的激光器 以快速成像速率跟踪单个分子所需的信噪比， TF扩散和染色质动力学在亚秒的时间尺度上是困难的。此补充请求是为了升级 通过增加先进的方位TIRF，我们目前TIRF显微镜的HILO显微镜功能 模块、更高功率激光器和像素配准的MEMS单分子成像模块。作为单一 分子成像直接关系到我们所有的原始目标，这种增强的能力将告知我们所有的 建议的研究。此外，高速成像能力将使我们能够应用我们新开发的 研究转录因子迁移率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性质和细胞微环境的机械性质调节转录。 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.