Expansion microscopy of axonemal dyneins in islet primary cilia

Abstract

ABSTRACTPrimary cilia are sensory organelles that mediate critical cellular functions yet are difficult to visualize because of their small size and low abundance. Expansion microscopy (ExM) is an imaging method that physically expands biological specimens using a swellable hydrogel, facilitating downstream imaging of small cellular structures. In this study, we apply ExM with confocal imaging to probe axonemal dynein expression in murine and human pancreatic islets. Innovations include an updated ExM workflow adapted from 2D cultured cells to 3D tissue optimized for intact pancreatic islets, and demonstration of axonemal dynein protein expression in islet primary cilia and centrioles. Four dynein subunits were consistently detected in both expanded and nonexpanded samples across species, including DNAI1, DNAH5, DNAH11, and DNALI1, where DNAI1 expression was the most readily detectable and seen to exhibit a distinct ring-like symmetry on super-resolution imaging. We discuss technical issues contributing to ExM success, including antibody performance across ExM and conventional sample preparation and imaging protocols, and the need to validate findings using gene knockdowns and controls. We conclude that ExM is a useful approach for identifying ciliary proteins in pancreatic islet tissue and may be broadly applicable to studying protein composition of other organelles.