Stress Granules In Cells Research Articles

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells

BackgroundThe eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown.MethodsSUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6–SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1–GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student’s t-test, or one-way or two-way analysis of variance (ANOVA).ResultsWe found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells.ConclusionsSUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.Graphical

The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

Development of a fluorescent chemical probe with the ability to visualize nascent phase-separated stress granules

Stress granules (SGs) are a fascinating type of membrane-less organelle that form through liquid-liquid phase separation in eukaryotic cells. Our previous investigations have been instrumental in the development of the first small-molecule fluorescent probe, named TASG, which can selectively identify SGs. However, the practical utility of TASG for monitoring SG dynamics in live cells has been hindered by the difficulty in visualizing small nascent SGs. To overcome this obstacle, the present study systematically modified the structure of TASG, with the aim of discovering fluorescent chemical probes that exhibit superior performance. Among the evaluated candidates, TASG-8 exhibited the most promising ability to image SGs. Importantly, TASG-8 possessed a critical advantage over TASG, in that it could effectively label small nascent SGs. The structure-activity relationship of the candidates was analyzed. Additionally, the underlying mechanism responsible for this enhancement was extensively explored. Collectively, our research presents a rational strategy for the development of innovative fluorescent probes that selectively target SGs, and introduces a powerful new tool, TASG-8, which has the potential to facilitate more comprehensive and efficient investigations into the functions of SGs in cells.

RNA helicase DDX6 and scaffold protein GW182 in P-bodies promote biogenesis of stress granules.

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.

Stress granules inhibit endoplasmic reticulum stress-mediated apoptosis during hypoxia-induced injury in acute liver failure.

Stress granules (SGs) could be formed under different stimulation to inhibit cell injury. To investigate whether SGs could protect hepatocytes from hypoxia-induced damage during acute liver failure (ALF) by reducing endoplasmic reticulum stress (ERS) mediated apoptosis. The agonist of SGs, arsenite (Ars) was used to intervene hypoxia-induced hepatocyte injury cellular model and ALF mice models. Further, the siRNA of activating transcription factor 4 (ATF4) and SGs inhibitor anisomycin was then used to intervene in cell models. With the increase of hypoxia time from 4 h to 12 h, the levels of HIF-1α, ERS and apoptosis gradually increased, and the expression of SGs marker G3BP1 and TIA-1 was increased and then decreased. Compared with the hypoxia cell model group and ALF mice model, the levels of HIF-1α, apoptosis and ERS were increased in the Ars intervention group. After siRNA-ATF4 intervention, the level of SGs in cells increased, and the levels of HIF-1α, ERS and apoptosis decreased. Compared with the siRNA-ATF4 group, the levels of G3BP1 in the siRNA-ATF4+anisomycin group were decreased, and the levels of HIF-1α, ERS and apoptosis were increased. Moreover, compared with the ALF group, the degree of liver injury and liver function, the levels of HIF-1α, ERS and apoptosis in the Ars intervention group were decreased, the level of SGs was increased. SGs could protect hepatocytes from hypoxia-induced damage during ALF by reducing ERS-mediated apoptosis.

Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Polyubiquitin effects on phase transitions of shuttle protein UBQLN2

ALS‐linked ubiquitin‐binding shuttle protein UBQLN2 mediates crosstalk between proteasomal degradation and autophagy pathways, which are thought to be controlled by interactions involving K48‐linked and K63‐linked polyubiquitin chains, respectively. We previously showed that UBQLN2 is recruited to stress granules in cells and undergoes liquid‐liquid phase separation (LLPS) in vitro. Interactions with ubiquitin or multivalent K48‐linked chains inhibit UBQLN2 LLPS. Here, we show that other multivalent chains of longer lengths, including K63‐ and M1‐linked chains and a designed tetrameric ubiquitin construct that mimics a multi‐monoubiquitinated substrate, significantly enhanced UBQLN2 LLPS over a wide range of Ub:UBQLN2 ratios. With nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in polyUb chain conformations, but not in affinities between polyUb chains and UBQLN2. Using microscopy and turbidity assay experiments, we obtained multi‐component phase diagrams to demonstrate that compact K11 and K48‐linked Ub4 inhibit UBQLN2 LLPS whereas extended K63 and M1‐linked Ub4 stabilize UBQLN2 LLPS. Increasing chain flexibility and accessibility to the ubiquitin binding surface enables a switch between homotypically‐driven to partially heterotypically‐driven UBQLN2 phase separation. These observations provide mechanistic insights into how UBQLN2 could differentiate between proteasomal degradation and autophagy pathways via LLPS.

Zinc enhances liquid-liquid phase separation of Tau protein and aggravates mitochondrial damages in cells

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer's disease. Liquid-liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregation can then be enhanced by zinc. However, it is unclear whether zinc modulates the formation of Tau stress granules in cells. We herein report that zinc promotes the formation of stress granules containing a pathological mutant ΔK280 of full-length human Tau. Furthermore, zinc promotes LLPS of ΔK280 of full-length Tau, shifting the equilibrium phase boundary to a lower protein concentration, and modulates the liquid nature of droplets formed by this pathological mutation. Zinc also promotes pathological phosphorylation of ΔK280 in neuronal cells, and aggravates mitochondrial damage and elevates reactive oxygen species production induced by Tau aggregation. Importantly, we show that treatment of cells with zinc increases the interaction between full-length Tau and G3BP1 inside stress granules to promote the formation of Tau filaments and increase Tau toxicity in neuronal cells. Collectively, these results demonstrate how Tau condensation and mitochondrial damages induced by Tau aggregation are enhanced by zinc to deteriorate the pathogenesis of Alzheimer's disease, bridging the gap between Tau LLPS and aggregation in neuronal cells.

RBM24 is localized to stress granules in cells under various stress conditions

Stress granules (SGs) are formed when untranslated messenger ribonucleoproteins (mRNPs) accumulate in cells under stress, and are thought to minimize stress-induced damage and promote cell survival. RBM24 (RNA-binding motif protein 24) is an RNA-binding protein that plays pivotal roles in regulating the stability or translation initiation of target mRNAs as well as alternative splicing of target pre-mRNAs. Its important physiological functions are highlighted by the fact that Rbm24 knockout mice or zebrafish suffer from dysfunction of heart, eye, and inner ear. Here we show that RBM24 is recruited into SGs under various stress conditions, suggesting that it might protect its target RNAs in cells under stress. However, SG formation is unaffected when Rbm24 expression is down-regulated. Nevertheless, RBM24 overexpression in cultured cells is sufficient to induce SG formation, suggesting that RBM24 might play an important role in SG formation. In conclusion, our present work reveals that RBM24 is a SG component, which implies that RBM24 could protect its target mRNAs in stressed cells.

African Swine Fever Virus K205R Induces ER Stress and Consequently Activates Autophagy and the NF-κB Signaling Pathway.

African swine fever virus (ASFV) is responsible for enormous economic losses in the global swine industry. The ASFV genome encodes approximate 160 proteins, most of whose functions remain largely unknown. In this study, we examined the roles of ASFV K205R in endoplasmic reticulum (ER) stress, autophagy, and inflammation. We observed that K205R was located in both the cytosolic and membrane fractions, and formed stress granules in cells. Furthermore, K205R triggered ER stress and activated the unfolded protein response through activating the transcription factor 6, ER to nucleus signaling 1, and eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3/PERK) signaling pathways. Moreover, K205R inhibited the serine/threonine kinase 1 and the mechanistic target of the rapamycin kinase signaling pathway, thereby activating unc-51 like autophagy activating kinase 1, and hence autophagy. In addition, K205R stimulated the translocation of P65 into the nucleus and the subsequent activation of the nuclear factor kappa B (NF-κB) signaling pathway. Inhibition of ER stress with a PERK inhibitor attenuated K205R-induced autophagy and NF-κB activation. Our data demonstrated a previously uncharacterized role of ASFV K205R in ER stress, autophagy, and the NF-κB signaling pathway.

Mechanistic insights into the promotion or inhibition of phase separation by polyubiquitin chains of different linkages

ALS-linked ubiquitin-binding shuttle protein UBQLN2 mediates crosstalk among several protein quality control pathways, including proteasomal degradation and autophagy that are likely mediated by interactions with K48-linked and K63-linked polyubiquitin chains, respectively. We recently showed that UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Surprisingly, other multivalent chains of longer lengths, including K63- and M1-linked chains and a designed tetrameric ubiquitin construct, significantly enhanced UBQLN2 LLPS by increasing the volume, but not the concentration, of the dense phase.

Grouper TIA-1 functions as a crucial antiviral molecule against nervous necrosis virus infection

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.

Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability.

Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

Lysine/RNA-interactions drive and regulate biomolecular condensation

Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer’s disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid–liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation.

ALS‐linked Mutations Modulate Ubiquilin‐2 Liquid‐Liquid Phase Separation

Cytoplasmic protein inclusions in post‐mortem motor neurons are a pathological hallmark of amyotrophic lateral sclerosis (ALS). Inclusions are hypothesized to arise from persistent stress granules. Stress granules are stress‐induced cytosolic ribonucleoprotein assemblies that form via liquid‐liquid phase separation (LLPS). They can dissipate when stress is removed; however, disease‐linked mutations in their protein components can disrupt stress granule dynamics and make them persist in cells. Persistent stress granules are hypothesized to accumulate and mature into cytoplasmic inclusions. Stress granule maintenance is regulated by the protein quality control (PQC) system. One of the PQC members is an ALS‐linked protein called ubiquilin‐2 (UBQLN2), which acts as a proteasomal shuttle factor and mediates protein degradation through the ubiquitin‐proteasome system and autophagy. Importantly, both wild‐type (WT) and mutant UBQLN2 have been found in inclusions in ALS patients. We previously demonstrated that UBQLN2 is recruited to stress granules in cells under different stress conditions and undergoes reversible LLPS in vitro under physiological conditions. UBQLN2 LLPS, a process in which proteins demix into liquid‐like droplets in solution, is driven by weak, multivalent interactions and is eliminated upon ubiquitin (Ub) binding. Many ALS‐linked mutations in UBQLN2 cluster to the proline‐rich Pxx region, which is unique to UBQLN2 along the other ubiquilin paralogs in humans. We hypothesized that the ALS‐linked mutations perturb UBQLN2 LLPS, alter droplet morphology, and that these mutants might respond to Ub binding differently from wild‐type (WT) UBQLN2. To probe the effects of ALS‐linked mutations on UBQLN2 LLPS, we used turbidity spectrophotometric assays to monitor UBQLN2 droplet formation and differential interference contrast microscopy to characterize droplet morphology. We used UBQLN2 450–624 (450C), the minimum length construct that undergoes LLPS, as a control, and examined proteins harboring 11 individual point mutations in the 450C background, T487I, A488T, P497H, P497L, P497S, P500S, P506A, P506S, P506T, P509S, and P525S. We found that a subset of ALS‐linked mutations (P506S, P506T, P506A, T487I, P497L, P497H, and P497S) significantly decreases the lower critical solution temperature (LCST) and thereby promotes UBQLN2 LLPS at lower temperatures. These mutations also promote LLPS over a larger temperature range. Five mutants within this subset (P506A, T487I, P497L, P497H, and P497S) form amorphous, gel‐like droplets or even aggregates. Ub clears WT and mutant UBQLN2 droplets and aggregates on a similar timescale.Support or Funding Information1. ALS Association grants 17‐IIP‐369 and 18‐IIP‐400 2. Syracuse University Meredith Scholar Award 3. Syracuse University Renée Crown University Honors Program Lynne Parker Award 4. Syracuse University Biology DepartmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Context‐dependent and Disease‐specific Diversity in Stress Granules Formed from Pre‐existing Protein Interactions

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration‐linked mutations, we used APEX proximity labeling, mass spectrometry and immunofluorescence to identify ~150 previously unknown human SG components. SG proteins form a dense interaction network in unstressed cells, facilitating rapid coalescence into larger SGs. About 20% of SG diversity is stress‐ or cell type‐dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition and subcellular distribution of SGs in cells from ALS patients. Using three Drosophila ALS/FTD models, we identify ~30 SG‐associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify ∼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity invivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.

Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly.

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

Involvement of endoplasmic reticulum stress in TDP-43-linked neurodegenerative disease

Phosphorylated and ubiquitinated TAR DNA binding protein 43 (TDP-43) pathology is the neurodegenerative hallmark of the vast majority of amyotrophic lateral sclerosis (ALS) and approximately half of all frontotemporal lobar degeneration (FTLD) cases. An RNA/DNA-binding protein with diverse roles in the nucleus, TDP-43 accumulates in the cytoplasm of affected neurons and glia in disease, indicating that both a loss of normal nuclear function and additional gain of toxicity due to increased cytoplasmic presence likely occur in these TDP-43 proteinopathies. Abundance and splicing of hundreds of transcripts are affected by dysfunction of TDP-43, making identification of the key pathways involved in disease pathogenesis a difficult task. However, endoplasmic reticulum (ER) stress and activation of the unfolded protein response have been described in sporadic ALS patient tissues as well as in cell and animals models of widely-studied superoxide dismutase 1 (SOD1) mutation-linked ALS. These responses to misfolded proteins have more recently been demonstrated in models of TDP-43linked disease, and modulation of ER stress, which is intimately associated with oxidative stress and formation of stress granules in cells, could therefore represent one avenue for potential amelioration of ALS and FTLD. This review summarizes recent findings describing the involvement of ER stress in TDP-43 proteinopathies.

Aspergillus oryzae AoSO Is a Novel Component of Stress Granules upon Heat Stress in Filamentous Fungi

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi.The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.