A correlative bioimaging force and multiomics platform for skin cell mechanosensing identification for new responsive proteins and the effects of agin

从单细胞迁移到组织发育和疾病的生物学，外部机械力和细胞外基质硬度在调节细胞行为方面非常重要。对动物细胞在外部动态定向拉伸下的研究揭示了包括细胞骨架重组在内的新的机械生物学反应。尽管已经确定了一些分子，但这些细胞重排还没有在拉伸过程中以高动态和特殊分辨率进行研究，据我们所知，高分辨率成像还没有与其他组学方法直接相关，也没有被用来理解细胞衰老。我们开发了一种显微镜拉伸系统，该系统能够对单向拉伸的皮肤细胞进行高分辨率荧光显微镜观察，同时结合活动力测量和精确的应变控制。使用这个系统，我们将成像处于拉伸状态的细胞，然后从这些细胞群体中分离蛋白质和RNA进行下游蛋白质组和转录分析，使我们不仅能够观察到新的精细动态反应，而且可以将其与蛋白质表达的变化相协调，以确定介导反应的蛋白质以及这些反应如何依赖于施加的力、细胞的整体性质和细胞的年龄。因此，该项目的总体目标是将单个高分辨率细胞特征(如细胞骨架、细胞连接和细胞外基质)与蛋白质组和转录分析以及细胞在拉伸过程中的机械响应/特性联系起来。这一独特的综合数据集资源可以被挖掘出来，在年轻和老年细胞中寻找标记和掌握细胞机械应力的调节器。这些相同的相关数据集也可以产生+/-生物活性物质，以确定哪些响应成分因特定的干预而减弱，从而将细胞生物学、蛋白质组学和转录组分与产品功效和作用模式联系起来。总体而言，该项目将有助于我们在分子和细胞水平上了解皮肤细胞如何对机械刺激做出反应。主要目标是：1)皮肤细胞(成纤维细胞和角质形成细胞，包括原代细胞)对机械应力的细胞反应的生物成像，包括以不同速率拉伸或周期性拉伸。2)使用定量蛋白质组学(SWATH)和RNA测序生成相关的蛋白质组和转录组数据集3)分析已知影响细胞机械性能的化合物和我们的工业合作伙伴感兴趣的选定化合物，以及已知影响体内皮肤结构特性的化合物。这将突出潜在的作用模式，并确定结构变化，这些变化可以与细胞特性的改变联系在一起4)了解老化的机械反应。部署上述技术来生成匹配的老化数据集(成像/强制/蛋白质组/转录组学)，我们可以询问该数据集，以确定蛋白质或转录本与老化细胞中看到的不同细胞反应之间的潜在联系。

External mechanical forces and ECM stiffness are important in regulating cell behaviour, from single cell migration to the biology of tissue development and disease. Studies of animal cells under external dynamic directional stretch have revealed novel mechanobiological responses including cytoskeletal reorganization. Although some molecular players have been identified these cellular rearrangements have not been studied at high dynamic and special resolution during stretch and, to our knowledge, high resolution imaging has not be directly correlated with other omics approaches or utilised to understand cellular ageing. We have developed an on-microscope stretch system which enables high resolution fluorescent microscopy of skin cells undergoing unidirectional stretch whilst incorporating live force measurements and accurate control of strain. Using this system we will image cells undergoing stretch and then isolate protein and RNA from these cell populations for downstream proteomic and transcriptomic analysis, allowing us to not only observe novel fine dynamic responses but coordinate this with the changes in protein expression to identify the proteins mediating the responses and how these responses depend on the force applied, the bulk properties of the cells, and the age of the cells. Thus, the overall objective of this project is to link individual high resolution cellular features (such as the cytoskeleton, cell junctions, and ECM) to proteomic and transcriptomic analysis and the mechanical response / properties of cells during stretch. This unique resource of integrated datasets can be mined for markers and master regulators of cellular mechanical stress in both young and old cells. These same correlated datasets can also be produced +/- bioactives to identify which responsive components are attenuated by particular interventions, linking cell biology, proteomics and transcriptomics to product efficacy and mode of action. Overall the project will contribute to our understanding of how skin cells respond to mechanical stimuli at the molecular and cellular level. The key objectives are: 1) Bioimaging of cellular responses of skin cells (fibroblasts and keratinocytes, including primary cells, to mechanical stress including stretch at different rates or cyclical stretching. 2) Generation of correlated proteomic and transcriptomic datasets using quantitative proteomics (SWATH) and RNA sequencing 3) Analysing compounds known to affect mechanical properties of cells and selected compounds of interest to our industrial partner, as well as compounds known to impact skin structural properties in vivo. This will highlight potential modes of action and identify structural changes which can be linked to modification of cell properties 4) Understanding the mechanical responses to ageing. Deploy the techniques described above to generate a matched dataset for ageing (Imaging/force/proteome/transcriptomics) which we can interrogate to identify potential links between protein or transcript and differing cellular responses seen in aged cells.