Biophysical basis of single cell force spectroscopy

从历史上看，免疫学研究一直是以蛋白质为中心的努力，信号事件通常被定义为多肽的生化变化。生物物理因素在这一计划中的作用基本上被忽视了。在我们上一次NSERC的支持下，我们使用了基于原子力显微镜(AFM)的单细胞力谱(SCFS)来阐明由细胞膜上的生物物理变化驱动的几个重要的免疫学事件。我们发现，颗粒物质以相当强的强度连接表面的脂质部分，导致双层膜的重排。这种脂质分选聚集信号分子，并触发吞噬和树突状细胞激活。因此，我们以前的工作将力和物理干扰的概念引入了免疫激活领域。在这项提议中，我们大大推进了这一概念，并提出免疫系统一直处于生物物理因素的调节之下。我们提供的证据表明，调节性T细胞是一组对控制自身免疫至关重要的CD4+T细胞，它与树突状细胞强烈结合，并抑制后者启动不必要的免疫激活的能力。我们还发现，T细胞和树突状细胞之间的稳态相互作用调节了系统中外周淋巴的体积或总免疫细胞的数量。也就是说，这两种细胞之间的物理结合提供了一个涓滴信号来控制免疫细胞的扩张和收缩。我们建议使用原子力显微镜和光子力显微镜来进一步研究这些相互作用的基本参数和与这些生物物理变化相关的信号事件。这项工作的结果将在免疫学研究中翻开新的一页，并将生物物理学引入免疫调节的一个重要方面。由于研究主题对公共卫生非常重要，用于开展这项工作的技术符合NSERC的核心使命，我们相信在未来几年在这里的投资将是非常有回报的。

Historically, immunological research has been a protein-centric endeavor, with signaling events often defined as biochemical changes of polypeptides. How biophysical factors play into this scheme has been mostly overlooked. Funded by our last NSERC support, we have used atomic force microscopy (AFM)-based single cell force spectroscopy (SCFS) to elucidate several important immunological events that are driven by biophysical changes at the cell membrane. We have found that particulate substances ligate surface lipid moieties with substantial intensity, causing rearrangement of the bilayer membrane. This lipid sorting aggregates signaling molecules and triggers phagocytosis and dendritic cell activation. Our previous work therefore introduces the concept of force and physical disturbance into the realm of immune activation. In this proposal, we substantially move this concept forward and suggest that the immune system is constantly under the regulation of biophysical factors. We provide evidence that regulatory T cells, a group of CD4+ T cells essential to control autoimmunity, bind strongly to dendritic cells and suppress the latter’s ability to mount unwanted immune activation. We also show that peripheral lymphoid volume or the number of total immune cells in the system is regulated by steady state interaction between T cells and dendritic cells. Namely the physical binding between these two cell types provides a trickling signal to control the expansion and contraction of immune cells. We propose to use AFM and photonic force microscopy to further study the essential parameters of these interactions and signaling events associated with these biophysical changes. The outcomes of this work will open a new page in immunological research, and introduce biophysics as an essential aspect of immune regulation. Because the topic of research is of great importance in public health and technologies used to carry the work are in line with the central mission of NSERC, we believe that the investment here will be highly rewarding in years to come.