Department of Biology, Temple University, Philadelphia, PA, United States.
Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, United States.
Methods Cell Biol. 2023;176:181-197. doi: 10.1016/bs.mcb.2022.12.016. Epub 2023 Jan 27.
The primary cilium is a surface exposed organelle found in eukaryotic cells that functions to decode a variety of intracellular signals with significant implications in human developmental disorders and diseases. It is therefore highly desirable to obtain in vivo information regarding the dynamic processes occurring within the primary cilium. However, current techniques are limited by either the physical limitations of light microscopy or the static nature of electron microscopy. To overcome these limitations, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed to obtain dynamic in vivo information in subcellular organelles such as cilia and nuclear pore complexes using single-molecule super-resolution light microscopy with a spatiotemporal resolution of 10-20nm and 0.4-2ms. Three-dimensional (3D) structural and dynamic information in these organelles can be further obtained through a post-processing 2D-to-3D transformation algorithm. Here we present a modular step-by-step protocol for studying primary cilium signaling dynamics, including Intraflagellar transport (IFT) via IFT20 and somatostatin g-protein-coupled receptor activity via SSTR3.
纤毛是真核细胞表面暴露的细胞器，可解码多种细胞内信号，对人类发育障碍和疾病有重要意义。因此，非常希望获得关于初级纤毛内发生的动态过程的体内信息。然而，目前的技术受到光学显微镜的物理限制或电子显微镜的静态性质的限制。为了克服这些限制，单点边缘激发亚衍射（SPEED）显微镜被开发出来，使用单分子超分辨率荧光显微镜以 10-20nm 的时空分辨率和 0.4-2ms 的时间分辨率，在纤毛和核孔复合物等亚细胞细胞器中获得动态的体内信息。通过二维到三维转换算法的后处理，可以进一步获得这些细胞器的三维结构和动态信息。在这里，我们提出了一个用于研究初级纤毛信号转导动力学的模块化分步方案，包括通过 IFT20 的鞭毛内运输（IFT）和通过 SSTR3 的生长抑素 G 蛋白偶联受体活性。
