DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells.-猫眼课题宝

DOI：

10.3791/62348

发表时间：

2021-03-20

期刊：

Journal of visualized experiments : JoVE

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

Ma R;Kellner AV;Hu Y;Deal BR;Blanchard AT;Salaita K

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8819630/

在两个相邻细胞之间的连接处以及在细胞与细胞外基质之间的连接处传递的机械力对于调节从发育到免疫学的许多过程至关重要。因此，开发在分子尺度上研究这些力的工具至关重要。我们的团队开发了一套分子张力传感器，以量化和可视化细胞产生的力并传递到特定的配体。最敏感的一类分子张力传感器由核酸茎环发夹组成。这些传感器使用荧光团-猝灭剂对来报告在力的作用下DNA发夹的机械延伸和展开。DNA发夹张力传感器的一个挑战是它们是可逆的，在张力终止时快速发夹重折叠，因此难以记录瞬时力。在这篇文章中，我们描述了用于制备DNA张力传感器的协议，该DNA张力传感器可以被“锁定”并防止重折叠以实现机械信息的“存储”。这允许记录高度瞬时的皮牛顿力，其随后可以通过添加移除锁定的互补核酸而被“擦除”。这种在实时张力映射和机械信息存储之间切换的能力揭示了T细胞通常作为其免疫功能的一部分使用的微弱，短暂和不太丰富的力量。本文描述了一个详细的协议，使用DNA为基础的张力探针图像的受体部队所施加的免疫细胞。这种方法可以实时绘制>4.7pN的受体力，并且可以随时间积分力。

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be “locked” and prevented from refolding to enable “storing” of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently “erased” by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions. This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces >4.7pN in real-time and can integrate forces over time.