Formation of the NLRP3 inflammasome inhibits stress granule assembly by multiple mechanisms.

Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key 'common' step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.

Stress granule formation inhibits stress-induced apoptosis by selectively sequestering executioner caspases.

Cytoplasmic stress granules (SGs) are phase-separated membrane-less organelles that form in response to various stress stimuli. SGs are mainly composed of non-canonical stalled 48S preinitiation complexes. In addition, many other proteins also accumulate into SGs, but the list is still incomplete. SG assembly suppresses apoptosis and promotes cell survival under stress. Furthermore, hyperformation of SGs is frequently observed in various human cancers and accelerates tumor development and progression by reducing stress-induced damage of cancer cells. Therefore, they are of clinical importance. However, the precise mechanism underlying SG-mediated inhibition of apoptosis remains ill-defined. Here, using a proximity-labeling proteomic approach, we comprehensively analyzed SG-resident proteins and identified the executioner caspases, caspase-3 and -7, as SG components. We demonstrate that accumulation of caspase-3/7 into SGs is mediated by evolutionarily conserved amino acid residues within their large catalytic domains and inhibits caspase activities and consequent apoptosis induced by various stresses. Expression of an SG-localization-deficient caspase-3 mutant in cells largely counteracted the anti-apoptotic effect of SGs, whereas enforced relocalization of the caspase-3 mutant to SGs restored it. Thus, SG-mediated sequestration of executioner caspases is a mechanism underlying the broad cytoprotective function of SGs. Furthermore, using a mouse xenograft tumor model, we show that this mechanism prevents cancer cells from apoptosis in tumor tissues, thereby promoting cancer progression. Our results reveal the functional crosstalk between SG-mediated cell survival and caspase-mediated cell death signaling pathways and delineate a molecular mechanism that dictates cell-fate decisions under stress and promotes tumorigenesis.

Caspase 3-specific cleavage of MEK1 suppresses ERK signaling and sensitizes cells to stress-induced apoptosis.

Proper regulation of apoptotic cell death is crucial for normal development and homeostasis in multicellular organisms and is achieved by the balance between pro-apoptotic processes, such as caspase activation, and pro-survival signaling, such as extracellular signal-regulated kinase (ERK) activation. However, the functional interplay between these opposing signaling pathways remains incompletely understood. Here, we identified MAPK/ERK kinase (MEK) 1, a central component of the ERK pathway, as a specific substrate for the executioner caspase-3. During apoptosis, MEK1 is cleaved at an evolutionarily conserved Asp282 residue in the kinase domain, thereby losing its catalytic activity. Gene knockout experiments showed that MEK1 cleavage was mediated by caspase-3, but not by the other executioner caspases, caspase-6 or -7. Following exposure of cells to osmotic stress, elevated ERK activity gradually decreased, and this was accompanied by increased cleavage of MEK1. In contrast, the expression of a caspase-uncleavable MEK1(D282N) mutant in cells maintained stress-induced ERK activity and thereby attenuated apoptotic cell death. Thus, caspase-3-mediated, proteolytic inhibition of MEK1 sensitizes cells to apoptosis by suppressing pro-survival ERK signaling. Furthermore, we found that a RASopathy-associated MEK1(Y130C) mutation prevented this caspase-3-mediated proteolytic inactivation of MEK1 and efficiently protected cells from stress-induced apoptosis. Our data reveal the functional crosstalk between ERK-mediated cell survival and caspase-mediated cell death pathways and suggest that its dysregulation by a disease-associated MEK1 mutation is at least partly involved in the pathophysiology of congenital RASopathies.

Involvement of clusterin expression in the refractory response of pancreatic cancer cells to a MEK inhibitor.

Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.

ERK-mediated NELF-A phosphorylation promotes transcription elongation of immediate-early genes by releasing promoter-proximal pausing of RNA polymerase II.

Growth factor-induced, ERK-mediated induction of immediate-early genes (IEGs) is crucial for cell growth and tumorigenesis. Although IEG expression is mainly regulated at the level of transcription elongation by RNA polymerase-II (Pol-II) promoter-proximal pausing and its release, the role of ERK in this process remains unknown. Here, we identified negative elongation factor (NELF)-A as an ERK substrate. Upon growth factor stimulation, ERK phosphorylates NELF-A, which dissociates NELF from paused Pol-II at the promoter-proximal regions of IEGs, allowing Pol-II to resume elongation and produce full-length transcripts. Furthermore, we found that in cancer cells, PP2A efficiently dephosphorylates NELF-A, thereby preventing aberrant IEG expression induced by ERK-activating oncogenes. However, when PP2A inhibitor proteins are overexpressed, as is frequently observed in cancers, decreased PP2A activity combined with oncogene-mediated ERK activation conspire to induce NELF-A phosphorylation and IEG upregulation, resulting in tumor progression. Our data delineate previously unexplored roles of ERK and PP2A inhibitor proteins in carcinogenesis.

Enhanced phosphorylation of c-Jun by cisplatin treatment as a potential predictive biomarker for cisplatin response in combination with patient-derived tumor organoids.

Despite recent advances in sequencing technology and large-scale drug screenings employing hundreds of cell lines, the predictive accuracy of mutation-based biomarkers is still insufficient as a guide for cancer therapy. Therefore, novel types of diagnostic methods using alternative biomarkers would be highly desirable. We have hypothesized that sensitivity-specific changes in the phosphorylation of signaling molecules could be useful in this respect. Here, with the aim of developing a method for predicting the response of cancers to cisplatin using a combination of specific biomarker(s) and patient-derived tumor organoids (PDOs), we found that cisplatin-sensitive cell lines or PDOs showed enhanced phosphorylation of c-Jun (p-c-Jun) within 24 h after cisplatin treatment. We also compared the responses of 6 PDOs to cisplatin with the therapeutic effect of neoadjuvant chemotherapy (docetaxel/cisplatin/5-fluorouracil) in 6 matched patients. Mechanistically, the c-Jun induction was partly related to TNF signaling induced by cisplatin. Our data suggest that enhanced phosphorylation of c-Jun in response to cisplatin treatment could be a predictive biomarker for the efficacy of cisplatin in selected cancer patients.

Qualitative differences in disease-associated MEK mutants reveal molecular signatures and aberrant signaling-crosstalk in cancer.

Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes. By making use of such distinct characteristics of the MEK1 mutants, we identified cancer- and RASopathy-signature genes that may serve as diagnostic markers or therapeutic targets for these diseases. In particular, two AKT-inhibitor molecules, PHLDA1 and 2, are simultaneously upregulated by oncogenic ERK signaling, and mediate cancer-specific ERK-AKT crosstalk. The combined expression of PHLDA1/2 is critical to confer resistance to ERK pathway-targeted therapeutics on cancer cells. Finally, we propose a therapeutic strategy to overcome this drug resistance. Our data provide vital insights into the etiology, diagnosis, and therapeutic strategy of cancers and RASopathies.

Prohibitin-1 Contributes to Cell-to-Cell Transmission of Herpes Simplex Virus 1 via the MAPK/ERK Signaling Pathway.

Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. Ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae, whereas gE is conserved only in the Alphaherpesvirinae subfamily.IMPORTANCE Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via a downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses.

Stress-responsive MTK1 SAPKKK serves as a redox sensor that mediates delayed and sustained activation of SAPKs by oxidative stress.

Cells respond to oxidative stress by inducing intracellular signaling, including stress-activated p38 and JNK MAPK (SAPK) pathways, but the underlying mechanisms remain unclear. Here, we report that the MAP three kinase 1 (MTK1) SAPK kinase kinase (SAPKKK) functions as an oxidative-stress sensor that perceives the cellular redox state and transduces it into SAPK signaling. Following oxidative stress, MTK1 is rapidly oxidized and gradually reduced at evolutionarily conserved cysteine residues. These coupled oxidation-reduction modifications of MTK1 elicit its catalytic activity. Gene knockout experiments showed that oxidative stress-induced SAPK signaling is mediated by coordinated activation of the two SAPKKKs, MTK1 and apoptosis signal-regulating kinase 1 (ASK1), which have different time and dose-response characteristics. The MTK1-mediated redox sensing system is crucial for delayed and sustained SAPK activity and dictates cell fate decisions including cell death and interleukin-6 production. Our results delineate a molecular mechanism by which cells generate optimal biological responses under fluctuating redox environments.

MCRIP1 promotes the expression of lung-surfactant proteins in mice by disrupting CtBP-mediated epigenetic gene silencing.

Proper regulation of epigenetic states of chromatin is crucial to achieve tissue-specific gene expression during embryogenesis. The lung-specific gene products, surfactant proteins B (SP-B) and C (SP-C), are synthesized in alveolar epithelial cells and prevent alveolar collapse. Epigenetic regulation of these surfactant proteins, however, remains unknown. Here we report that MCRIP1, a regulator of the CtBP transcriptional co-repressor, promotes the expression of SP-B and SP-C by preventing CtBP-mediated epigenetic gene silencing. Homozygous deficiency of Mcrip1 in mice causes fatal respiratory distress due to abnormal transcriptional repression of these surfactant proteins. We found that MCRIP1 interferes with interactions of CtBP with the lung-enriched transcriptional repressors, Foxp1 and Foxp2, thereby preventing the recruitment of the CtBP co-repressor complex to the SP-B and SP-C promoters and maintaining them in an active chromatin state. Our findings reveal a molecular mechanism by which cells prevent inadvertent gene silencing to ensure tissue-specific gene expression during organogenesis.

Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis.

Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked ß-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, however, needs multiple and time-consuming steps if using conventional methods (e.g., immune-purification, mass spectrometric analysis). To overcome this drawback, we developed the following easy and rapid method for detection of O-GlcNAcylated proteins of interest. An O-GlcNAc affinity gel layer containing wheat germ agglutinin (WGA), a GlcNAc-specific lectin, selectively induces retardation of the mobility of O-GlcNAcylated proteins during electrophoresis. This WGA-layer thereby separates O-GlcNAcylated and non-modified forms of proteins, allowing the detection and quantification of the O-GlcNAcylation level of these proteins. This new method therefore provides qualitative and quantitative analysis of O-GlcNAcylated proteins in a relatively shorter time compared to conventional methods.

Downregulation of dual-specificity phosphatase 4 enhances cell proliferation and invasiveness in colorectal carcinomas.

It is widely accepted that aberrant activation of the Wnt signaling pathway is responsible for the development of precursor lesions of colorectal cancer (CRC). However, the molecular mechanisms involved in the process of progression from these precursor lesions to invasive lesions of CRC are not fully understood. Recently, we reported that constitutive activation of MAPK accompanied by downregulation of dual-specificity phosphatase 4 (DUSP4), a MAPK phosphatase, contributes to the progression of precursor lesions in the pancreas. In this study, we found that downregulation of DUSP4 was related to constitutive activation of ERKs in CRC cells. Restoration of DUSP4 resulted in inactivation of ERKs, leading to suppression of both proliferation and invasiveness, as shown by treatment with an MEK inhibitor. Furthermore, immunohistochemistry revealed that DUSP4 expression was upregulated in the superficial region of CRC tissue, whereas it was significantly downregulated in the deep region. In contrast, ERKs in the deep region were markedly hyperactivated compared to those in the superficial region. These results suggest that activation of the MAPK signaling pathway caused by downregulation of DUSP4 is responsible for progression of CRCs and would be a promising therapeutic target.

A guanine derivative as a new MEK inhibitor produced by Streptomyces sp. MK63-43F2.

Mitogen-activated protein kinase (MAPK) pathways that direct cellular responses are involved in various biological processes; the RAS-RAF-MEK-ERK pathway is one of the most important MAPK pathways. It is frequently activated in human malignant tumors such as melanomas, thyroid tumors and colorectal carcinomas. Therefore, targeting this pathway has been considered an attractive strategy for new anticancer drugs. In particular, MEK is a promising target because it is a kinase that directly phosphorylates ERK. We performed a screening to discover new MEK inhibitors, and found a guanine derivative produced by Streptomyces sp. MK63-43F2. This guanine derivative was identified to be 2-amino-4-methoxy-5-cyanopyrrolo[2,3-d]pyrimidine (1) through spectroscopic analysis. Compound 1 inhibited MEK1 kinase activity in an ATP-dependent manner and suppressed the phosphorylation of ERK in cancer cells and cell proliferation. Therefore, 1 might be a potent lead compound for new MEK inhibitors.The Journal of Antibiotics advance online publication, 6 September 2017; doi:10.1038/ja.2017.100.

WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins.

Post-translational modification with O-linked ß-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Detection and Functional Analysis of SUMO-Modified MEK.

Small ubiquitin-like modifier (SUMO) is a posttranslational protein modifier that binds target proteins covalently (protein sumoylation) and remarkably alters their functions. Protein sumoylation has been linked to various cellular functions such as cell division, DNA repair, and import of nuclear proteins. Thus, its dysregulation is implicated in diverse human diseases such as neurodegenerative disorders and cancers. We recently found that the kinase activity of MEK proteins, which function as central components of the ERK-MAPK cascade and amplify an extracellular proliferation signal, is negatively regulated by sumoylation. Moreover, the oncogenic activity of Ras is enhanced by the abrogation of MEK-sumoylation in cancer cells. Here, we describe several tools and techniques utilized for the elucidation of the properties of SUMO-MEK in our previous reports. We believe that these methods can be used as robust tools for investigating and understanding the biological roles of various SUMO-modified (sumoylated) proteins.

A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1.

MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126.

Genomic Loss of DUSP4 Contributes to the Progression of Intraepithelial Neoplasm of Pancreas to Invasive Carcinoma.

The progression from precursor lesions of pancreatic cancer, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN), to invasive disease is characterized by stepwise accumulation of genetic alterations. However, it remains unclear whether additional alterations are required for the progression of high-grade neoplasms to invasive pancreatic carcinoma. We compared the genomic profiles of paired noninvasive and invasive carcinoma tissues collected from patients with IPMN. We demonstrate that the frequency of genomic copy-number aberrations significantly increased during the course of invasion, and the loss of 8p11.22-ter was more often associated with invasive tissues. Expression profiling in pancreatic cancer cell lines with and without 8p11.22-ter revealed that DUSP4, an MAPK phosphatase, was significantly downregulated in cells lacking 8p11.22-ter as well as in invasive carcinomas due to genomic loss. Restoration of DUSP4 expression in pancreatic cancer cells significantly suppressed invasiveness and anoikis resistance via ERK inactivation. Accordingly, we found that blockade of ERK signaling by MEK inhibition was effective in an orthotopic xenograft model and significantly extended survival. Collectively, our findings demonstrate a genetic mechanism by which pancreatic precursor lesions progress to invasive carcinomas and highlight DUSP4 as a novel invasion suppressor that can be therapeutically exploited through manipulation of ERK signaling. Cancer Res; 76(9); 2612-25. ©2016 AACR.

TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis.

Cytoplasmic stress granules (SGs) are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of misfolded proteins, and that are formed in response to certain types of stress including ER stress. SG formation contributes to cell survival not only by suppressing translation but also by sequestering some apoptosis regulatory factors. Because cells can be exposed to various stresses simultaneously in vivo, the regulation of SG assembly under multiple stress conditions is important but unknown. Here we report that reactive oxygen species (ROS) such as H2O2 oxidize the SG-nucleating protein TIA1, thereby inhibiting SG assembly. Thus, when cells are confronted with a SG-inducing stress such as ER stress caused by protein misfolding, together with ROS-induced oxidative stress, they cannot form SGs, resulting in the promotion of apoptosis. We demonstrate that the suppression of SG formation by oxidative stress may underlie the neuronal cell death seen in neurodegenerative diseases.

Oscillation of p38 activity controls efficient pro-inflammatory gene expression.

The p38 MAP kinase signalling pathway controls inflammatory responses and is an important target of anti-inflammatory drugs. Although pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) appear to induce only transient activation of p38 (over ∼ 60 min), longer cytokine exposure is necessary to induce p38-dependent effector genes. Here we study the dynamics of p38 activation in individual cells using a Förster resonance energy transfer (FRET)-based p38 activity reporter. We find that, after an initial burst of activity, p38 MAPK activity subsequently oscillates for more than 8 h under continuous IL-1ß stimulation. However, as this oscillation is asynchronous, the measured p38 activity population average is only slightly higher than basal level. Mathematical modelling, which we have experimentally verified, indicates that the asynchronous oscillation of p38 is generated through a negative feedback loop involving the dual-specificity phosphatase MKP-1/DUSP1. We find that the oscillatory p38 activity is necessary for efficient expression of pro-inflammatory genes such as IL-6, IL-8 and COX-2.

Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly.

Stress granules (SGs) are non-membranous cytoplasmic aggregates of mRNAs and related proteins, assembled in response to environmental stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, chemicals (e.g. arsenite), and viral infections. SGs are hypothesized as a loci of mRNA triage and/or maintenance of proper translation capacity ratio to the pool of mRNAs. In brain ischemia, hippocampal CA3 neurons, which are resilient to ischemia, assemble SGs. In contrast, CA1 neurons, which are vulnerable to ischemia, do not assemble SGs. These results suggest a critical role SG plays in regards to cell fate decisions. Thus SG assembly along with its dynamics should determine the cell fate. However, the process that exactly determines the SG assembly dynamics is largely unknown. In this paper, analyses of experimental data and computer simulations were used to approach this problem. SGs were assembled as a result of applying arsenite to HeLa cells. The number of SGs increased after a short latent period, reached a maximum, then decreased during the application of arsenite. At the same time, the size of SGs grew larger and became localized at the perinuclear region. A minimal mathematical model was constructed, and stochastic simulations were run to test the modeling. Since SGs are discrete entities as there are only several tens of them in a cell, commonly used deterministic simulations could not be employed. The stochastic simulations replicated observed dynamics of SG assembly. In addition, these stochastic simulations predicted a gamma distribution relative to the size of SGs. This same distribution was also found in our experimental data suggesting the existence of multiple fusion steps in the SG assembly. Furthermore, we found that the initial steps in the SG assembly process and microtubules were critical to the dynamics. Thus our experiments and stochastic simulations presented a possible mechanism regulating SG assembly.