Department of Medicine, Washington University School of Medicine, Saint Louis, MO, United States.
Department of Mechanical Engineering and Materials Science, Washington University McKelvey School of Engineering, Saint Louis, MO, United States.
Front Endocrinol (Lausanne). 2022 Sep 23;13:1004136. doi: 10.3389/fendo.2022.1004136. eCollection 2022.
Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the environment. We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.
初级纤毛是伸向细胞间隙的细长细胞表面细胞器。在胰腺β细胞中，初级纤毛协调各种细胞反应，包括 G 蛋白偶联受体信号转导、钙离子内流和胰岛素分泌，以及在胰岛发育和分化中可能有许多未被充分认识的作用。为了研究纤毛在胰岛生物学中的功能，通过显微镜方法直接观察初级纤毛通常是必要的第一步。胰岛细胞中的纤毛丰度、分布和形态存在异质性，最好通过荧光显微镜观察，大多数研究人员都可以轻松获得这些工具。在这里，我们介绍了一组荧光成像方法，我们已经采用并优化了这些方法，用于观察小鼠和人类胰岛中的初级纤毛。这些方法包括使用固定胰岛和胰腺切片的传统共聚焦显微镜、使用纤毛靶向生物传感器和探针的活细胞成像、纤毛运动记录以及在环境中对初级纤毛波形的定量分析。我们讨论了我们方法的实际考虑因素和局限性，以及新的工具，以促进对胰腺胰岛中初级纤毛的观察。
