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 (MLOs). Examples of MLOs include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and post-translational 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 C. elegans can cause an inadvertent stress and trigger phase transitions of RNA binding proteins. We show this imaging-associated stress stimulates decondensation of multiple germ granule proteins, and condensation of several P-body proteins. Proteins within larger RNP 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 RNP granules of arrested oocytes may have distinct phases which we characterize in our companion paper.

Large RNP granules in Caenorhabditis elegans oocytes have distinct phases of RNA-binding proteins

“…We therefore also asked if MEX-3 condenses in response to elevated temperature in diakinesis oocytes. In contrast to the modest change in MEX-3 condensation observed during extended imaging in our companion paper ( Elaswad et al 2022 ), the majority of the MEX-3 protein that is normally dispersed throughout the oocyte cytosol appeared condensed into granules at the cortex or nuclear envelope ( Fig. 1, a and b ).…”

“…In a companion article ( Elaswad et al 2022 ), we showed that imaging-associated stress triggers opposite phase transition-responses of PGL-1 and MEX-3/CGH-1/MEG-3. These results suggested the possibility that PGL-1 is a liquid phase of the large RNP granules in arrested oocytes, while MEX-3, CGH-1 and MEG-3 may be gel-like.…”

“…The FRAP data for CGH-1 showed strongly dynamic behavior within large RNP granules. These data were somewhat surprising since large granules of CGH-1 in arrested oocytes were not sensitive to imaging-associated stress, nor strongly sensitive to elevated temperature or HD treatment ( Table 1 ; Elaswad et al 2022 ). CGH-1 may be an intermediate phase of large RNP granules, distinct from, and less liquid like than PGL-1 along the relative continuum of phase separation.…”

“…In this study, we build upon the findings in our companion article where we characterized an imaging-associated stress that differentially modulates the condensation of RNA-binding proteins in oocytes ( Elaswad et al 2022 ). The stress appears to be specifically attributed to the preparation of worms for imaging, which involves mounting them on agarose pads under a coverslip, and we show that inadvertent phase transitions can begin to occur if images are not captured within 10 min of sample preparation.…”

“…The stress appears to be specifically attributed to the preparation of worms for imaging, which involves mounting them on agarose pads under a coverslip, and we show that inadvertent phase transitions can begin to occur if images are not captured within 10 min of sample preparation. PGL-1 and the DEAD box helicase GLH-1 decondense in response to this imaging-associated stress, while the KH domain protein MEX-3, DEAD box helicase CGH-1, and Lsm protein CAR-1 condense into granules ( Elaswad et al 2022 ). This differential stress response suggested that RNA-binding proteins in large RNP granules of arrested oocytes may have distinct biophysical properties.…”