Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP’s widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C. elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment. [Display omitted] •The cuticle of Caenorhabditis elegans has long hampered fluorescent staining approaches•This cryosection method allows staining of hundreds of worms in a single experiment•Staining for polyphosphate reveals its presence in endo-lysosomal compartments•Polyphosphate levels are sensitive to food availability Caenorhabditis elegans are a widely used system to study basic biological phenomena. However, studies requiring fluorescently tagged molecules are restricted to genetically manipulated organisms or low-throughput and disruptive staining techniques, in part due to the presence of a tough cuticle. Labeling polyphosphate with known probes is not compatible with either of these options in a high-throughput setting. Thus, we describe a method to cryosection worms longitudinally and en masse to allow for staining with fluorescent probes with minimal chemical perturbation of the samples. Polyphosphate (polyP), a ubiquitous and highly conserved molecule, is present throughout all branches of life yet has mostly been studied in single cells. Quarles et al. describe a method to longitudinally section Caenorhabditis elegans, allowing high-throughput staining of worms using polyP-specific fluorescent probes. These studies establish C. elegans as a model for polyP research.