Processing bodies and Stress granules oscillate in Neuro 2a cells and mouse fibroblasts

PENAZZI LG; MALCOLM M; GARBARINO PICO E

Congreso; XXXIV Reunión Anual SAN; 2019

Sociedad Argentina de Investigaciones en Neurociencias

Processingbodies (PB) and Stress Granules (SG) are membraneless organelles involved inmRNA storage, processing and decay. Whereas PB are constitutive organelles, SGare assembled in response to stress. We have previously shown that SGs inducedby oxidative stress and PBs display daily changes in their number and size insynchronized NIH3T3 cultures. Here, in order to determine whether these changesare controlled by a biological clock, we analyzed SG and PB between 8h and 68hpostsynchronization with a higher sampling frequency. We induced SG formationby oxidative stress with sodium arsenite. We also studied the temporalexpression profile of p-eIF2α and eIF3 (factors involved in SG aggregation) byWestern blot. For this purpose N2a cells and fibroblasts were grown and thenarrested to prevent cell cycle progression. They were then synchronized andharvested at different times. We confirmed that cells were in quiescent stateby flow cytometry. We performed ICC with anti-eIF3 primary antibody, a known SGmarker, in NIH3T3 fibroblast and we used two PB markers: anti-DDX6/P54/RCK andanti-GE-1/HEDLS in double immunolabelling experiments in N2a cells. Metacyleanalysis revealed that both SG and PB were rhythmic for 2 cycles with periodsof about 24h for all variables analyzed (Number per field, Area and Signalintensity). The p-eIF2α and eIF3 proteins presented slight changes over timeand therefore would not be responsible for generating the changes observed inSGs.