Abstract
The survival of cells exposed to adverse environmental conditions entails various alterations in cellular function including major changes in the transcriptome as well as a radical reprogramming of protein translation. While in mammals this process has been extensively studied, stress responses in non-mammalian vertebrates remain poorly understood. One of the key cellular responses to many different types of stressors is the transient generation of structures called stress granules (SGs). These represent cytoplasmic foci where untranslated mRNAs are sorted or processed for re-initiation, degradation, or packaging into mRNPs. Here, using the evolutionarily conserved Y-box binding protein 1 (YB-1) and G3BP1 as markers, we have studied the formation of stress granules in zebrafish (D. rerio) in response to different environmental stressors. We show that following heat shock, zebrafish cells, like mammalian cells, form stress granules which contain both YB-1 and G3BP1 proteins. Moreover, zfYB-1 knockdown compromises cell viability, as well as recruitment of G3BP1 into SGs, under heat shock conditions highlighting the essential role played by YB-1 in SG assembly and cell survival. However, zebrafish PAC2 cells do not assemble YB-1-positive stress granules upon oxidative stress induced by arsenite, copper or hydrogen peroxide treatment. This contrasts with the situation in human cells where SG formation is robustly induced by exposure to oxidative stressors. Thus, our findings point to fundamental differences in the mechanisms whereby mammalian and zebrafish cells respond to oxidative stress.
Highlights
Stress caused by environmental insults or disease can disrupt cellular, tissue and organ homeostasis
As previously reported for mammalian cells[20], immunofluorescence staining of the PAC2 cell line using the C-ter Y-box binding protein 1 (YB-1) antibody, detected a predominantly cytoplasmic signal which was distributed in a fine, punctate pattern (Fig. 1a, left panel)
By immunofluorescence assays performed in PAC2 cells, the N-ter YB-1 antibody detected mainly a nuclear signal and in western blot analysis it clearly detected all the forms recognized by the C-ter antibody with the exception of the 50 kDa band that was only barely detectable (Fig. 1a, right side of the panels and Fig. 1b)
Summary
Stress caused by environmental insults or disease can disrupt cellular, tissue and organ homeostasis. Eukaryotes respond to detrimental conditions by activating a set of conserved processes that aim to re-establish cellular homeostasis This multifaceted response is critical for cell survival[1]. It is characterized by stress-dependent changes in the transcriptome and down-regulation of global translation[2]. In SGs, messengers are sequestered and regulated following stressful conditions They possess mRNA in a repressed state that may subsequently re-initiate translation in response to specific signals[7]. SG assembly appears to represent a highly conserved cellular strategy to minimize stress-related damage and promote cell www.nature.com/scientificreports/. In addition to saving anabolic energy by preventing the synthesis of housekeeping proteins, SGs promote cell survival by sequestering pro-apoptotic factors and promoting the translation of stress activated messengers such as BCL2 and ATF412–14. The composition of SG protein aggregates is well studied, their precise function remains unclear and how RNAs are sorted and regulated in SGs is still unknown[15,16,17]
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