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

细胞机械转导的补充请求 - 从免疫反应到转录调控

• 批准号: 10799068
• 负责人: Arpita Upadhyaya
• 金额: $ 24.94万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799068
• 关键词: Adhesions; Algorithms; Award; Binding; Biochemical; Breast; Cell Nucleus; Cell physiology; Cells; Chemicals; Chromatin; Color; Complement; Complex; Cues; Cytoskeleton; Diffusion; Disease Marker; Equipment; Exhibits; Fluorescence; Gel; Gene Expression; Gene Expression Regulation; Genomics; Glass; Histones; Hydrogels; Image; Imaging technology; Immune; Immune response; Lasers; Malignant Neoplasms; Mechanics; Mediating; Microscope; Microscopy; Movement; Noise; Optics; Pathway interactions; Receptor Signaling; Resolution; Risk Factors; Signal Transduction; Speed; Substrate Interaction; Thick; Tissues; Transcriptional Regulation; Visualization; cancer cell; cellular imaging; density; fluorescence microscope; imaging capabilities; imaging system; light scattering; mechanical force; mechanical properties; mechanical signal; mechanotransduction; molecular imaging; nanoscale; single molecule; transcription factor; tumor progression

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.

由粘附和信号传导受体介导的细胞与细胞和细胞与基质的相互作用是高度动态的和 受到细胞骨架运动的影响，在界面处施加大量机械力。细胞如何结合 执行特定功能的机械和生化信号尚不清楚。受此支持 MIRA 奖，我们正在研究机械线索如何传递到细胞核以调节基因表达 以功能适当的方式以及机械线索如何与组织特异性线索相互作用。这需要 能够进行多色单分子成像、具有高信噪比的成像系统 细胞核。高倾斜层压光学片 (HILO) 显微镜是一种成像技术，允许 用于以纳米级分辨率可视化细胞内部深处的单分子。单身的 转录因子迁移性和结合的分子成像产生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与这些染色质迁移模式结合的 TF 之间的关系。为了 为了研究细胞如何整合机械和化学信号，我们使用刚度可调的水凝胶 通过机械线索检查转录调节。而传统的全内反射 具有荧光 (TIRF) 功能的显微镜可适用于玻璃盖玻片上细胞的 HILO 成像， 由于厚度和多余的水凝胶（30-50 微米厚度）上的成像细胞提出了挑战 中间凝胶内的光散射。此外，在我们的设置限制内功率相对较低的激光器 以快速成像速率跟踪单分子所需的信噪比，从而进行分析 亚秒级时间尺度的 TF 扩散和染色质动力学很困难。此补充请求是升级 通过添加先进的方位 TIRF，我们当前的 TIRF 显微镜具有 HILO 显微镜功能 模块、更高功率的激光器和像素配准的多色单分子成像模块。作为单身 分子成像与我们所有最初的目标直接相关，这种增强的能力将告知我们所有 拟议的研究。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。我们的机械传感研究 以及细胞微环境的机械特性和转录的调节 潜在的途径将增进我们对乳腺癌和其他癌症的了解。

项目成果

Transcription Factor Dynamics: One Molecule at a Time.

转录因子动力学：一次一个分子。

• 发表时间: 2023-10-16
• 作者: Wagh, Kaustubh;Stavreva, Diana A;Upadhyaya, Arpita;Hager, Gordon L
• 通讯作者: Hager, Gordon L

A tug of war between filament treadmilling and myosin induced contractility generates actin rings.

肌丝跑步和肌球蛋白诱导的收缩性之间的拉锯战产生了肌动蛋白环。

• 发表时间: 2022-10-21
• 影响因子: 7.7
• 作者: Ni, Qin;Wagh, Kaustubh;Pathni, Aashli;Ni, Haoran;Vashisht, Vishavdeep;Upadhyaya, Arpita;Papoian, Garegin A
• 通讯作者: Papoian, Garegin A

Dynamic switching of transcriptional regulators between two distinct low-mobility chromatin states.

转录调节因子在两种不同的低迁移率染色质状态之间的动态切换。

• 发表时间: 2023-06-16
• 影响因子: 13.6
• 作者: Wagh, Kaustubh;Stavreva, Diana A;Jensen, Rikke A M;Paakinaho, Ville;Fettweis, Gregory;Schiltz, R Louis;Wüstner, Daniel;Mandrup, Susanne;Presman, Diego M;Upadhyaya, Arpita;Hager, Gordon L
• 通讯作者: Hager, Gordon L