Fluorescence imaging of beta cell primary cilia

Abstract

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 ex vivo 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.

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

“…Cilia across spatial scales were tracked by morphology and their identification aided by immunogold labeling of ciliary protein markers. Preservation of ciliary structures was excellent as most cilia axonemes remained intact, exhibiting similar lengths (4-10 µm) and diameters (200-300 nm at base) as previously characterized by us and others 9,11,14,15 . We found moderate magnification (20-35,000x) to give a good overview of the entire ciliary structure.…”

“…These primary antibodies were used in conjunction with standard gold-conjugated secondary antibodies specific for mouse and rabbit IgG. Table 1 lists the source and labeling conditions of all primary antibodies, typically used at 5-to 10-fold higher concentrations as for immunofluorescence imaging 8,[11][12][13] . Some but not all primary antibodies were tested in both mouse and human islets -species indicated in figures.…”

Scanning electron microscopy of human islet cilia

“…In human islets, primary cilia were first described in beta cells, in 1964 in a case of insulinoma (13), whereas validation in non-diseased islet tissues took decades to follow (14)(15)(16). More recently, islet primary cilia were found to have glucoseregulated motility, suggesting that primary cilia are not simply static sensory organelles (16,17). These findings have fueled growing interest in islet primary cilia in diabetes research, paralleled by an increasing need to understand the basic structure and composition of these cellular projections.…”

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

“…Besides Click-iT chemistry and SNAP/HALO tag technology, low-molecular weight reagents for labelling other cellular structures (primarily cytoskeleton—SiR and SPY probes; ref. 43 ) are available, though the spectrum of applications is limited. Our approach will benefit from the current effort to introduce other labelling strategies which aim to minimize the size of the labelling reagents and thus increase the permeability through Lowicryl resin, including detection with scFv antibody fragments, nanobodies 44 , 45 , proximity labelling with biotin 46 or DAB precipitation-based probes (reviewed in ref.…”