Imaging-associated stress causes divergent phase transitions of RNA-binding proteins in the Caenorhabditis elegans germ line

Abstract

One emerging paradigm of cellular organization of RNA and RNA-binding proteins is the formation of membraneless organelles. Examples of membraneless organelles include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and posttranslational modifications, as well as extracellular stresses, can stimulate the condensation of proteins into granules. For example, the assembly of stress granules induced by oxidative stress, osmotic stress, and heat stress has been well characterized in a variety of somatic cell types. In the germ line, similar stress-induced condensation of proteins occurs; however, less is known about the role of phase separation during gamete production. Researchers who study phase transitions often make use of fluorescent reporters to study the dynamics of RNA-binding proteins during live cell imaging. In this report, we demonstrate that common conditions of live-imaging Caenorhabditis elegans can cause an inadvertent stress and trigger phase transitions of RNA-binding proteins. We show that this imaging-associated stress stimulates decondensation of multiple germ granule proteins and condensation of several P-body proteins. Proteins within larger ribonucleoprotein granules in meiotically arrested oocytes do not appear to be as sensitive to the stress as proteins in diakinesis oocytes of young hermaphrodites, with the exception of the germ granule protein PGL-1. Our results have important methodological implications for all researchers using live-cell imaging techniques. The data also suggest that the RNA-binding proteins within large ribonucleoprotein granules of arrested oocytes may have distinct phases, which we characterize in our companion article.

Keywords: RNA-binding protein; RNP granule; condensation; live imaging; oogenesis; phase transition; stress.