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

PENNAZI GABRIELA L.; GARBARINO PICO EDUARDO; MALCOLM MELISA

Villa Carlos Paz

Congreso; XXXIV Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias; 2019

Sociedad Argentina de Investigación en Neurociencias

Processing bodies (PB) and Stress Granules (SG) are membraneless organelles involved in mRNA storage, processing and decay. Whereas PB are constitutive organelles, SG are assembled in response to stress. We have previously shown that SGs induced by oxidative stress and PBs display daily changes in their number and size in synchronized NIH3T3 cultures. Here, in order to determine whether these changes are controlled by a biological clock, we analyzed SG and PB between 8h and 68h postsynchronization with a higher sampling frequency. We induced SG formation by oxidative stress with sodium arsenite. We also studied the temporal expression profile of p-eIF2α and eIF3 (factors involved in SG aggregation) by Western blot. For this purpose N2a cells and fibroblasts were grown and then arrested to prevent cell cycle progression. They were then synchronized and harvested at different times. We confirmed that cells were in quiescent state by flow cytometry. We performed ICC with anti-eIF3 primary antibody, a known SG marker, in NIH3T3 fibroblast and we used two PB markers: anti-DDX6/P54/RCK and anti-GE-1/HEDLS in double immunolabelling experiments in N2a cells. Metacyle analysis revealed that both SG and PB were rhythmic for 2 cycles with periods of about 24h for all variables analyzed (Number per field, Area and Signal intensity). The p-eIF2α and eIF3 proteins presented slight changes over time and therefore would not be responsible for generating the changes observed in SGs.