Descripción: Stress granules are cytoplasmic mRNA-silencing foci that form transiently during the stress response. Stress granules harbor abortive translation initiation complexes and are in dynamic equilibrium with translating polysomes. Mammalian Staufen 1 (Stau1) is a ubiquitous double-stranded RNA-binding protein associated with polysomes. Here, we show that Stau1 is recruited to stress granules upon induction of endoplasmic reticulum or oxidative stress as well in stress granules induced by translation initiation blockers. We found that stress granules lacking Stau1 formed in cells depleted of this molecule, indicating that Stau1 is not an essential component of stress granules. Moreover, Stau1 knockdown facilitated stress granule formation upon stress induction. Conversely, transient transfection of Stau1 impaired stress granule formation upon stress or pharmacological initiation arrest. The inhibitory capacity of Stau1 mapped to the amino-terminal half of the molecule, a region known to bind to polysomes. We found that the fraction of polysomes remaining upon stress induction was enriched in Stau1, and that Stau1 overexpression stabilized polysomes against stress. We propose that Stau1 is involved in recovery from stress by stabilizing polysomes, thus helping stress granule dissolution.

Descripción: Stress granules (SGs) and P-bodies (PBs) are related cytoplasmic structures harboring silenced mRNAs. SGs assemble transiently upon cellular stress, whereas PBs are constitutive and are further induced by stress. Both foci are highly dynamic, with messenger ribonucleoproteins (mRNPs) and proteins rapidly shuttling in and out. Here, we show that impairment of retrograde transport by knockdown of mammalian dynein heavy chain 1 (DHC1) or bicaudal D1 (BicD1) inhibits SG formation and PB growth upon stress, without affecting proteinsynthesis blockage. Conversely, impairment of anterograde transport by knockdown of kinesin-1 heavy chain (KIF5B) or kinesin light chain 1 (KLC1) delayed SG dissolution. Strikingly, SG dissolution is not required to restore translation. Simultaneous knockdown of dynein and kinesin reverted the effect of single knockdowns on both SGs and PBs, suggesting that a balance between opposing movements driven by these molecular motors governs foci formation and dissolution. Finally, we found that regulation of SG dynamics by dynein and kinesin is conserved in Drosophila.

Descripción: Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHBsynthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins. © 2011, American Society for Microbiology.

Descripción: The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny. © 2012 Perez-Pepe et al.