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Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly.

Ohshima D, Arimoto-Matsuzaki K, Tomida T, Takekawa M, Ichikawa K - PLoS Comput. Biol. (2015)

Bottom Line: SGs were assembled as a result of applying arsenite to HeLa cells.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Mathematical Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

TIA-1 is required for the assembly of SG.(A) GFP alone or GFP-PRD was transiently transfected into COS-7 cells as indicated. After 48 h, the cells were treated with arsenite (0.5 mM) for 50 min. Endogenous eIF4E, a SG marker protein, was visualized by immunofluorescence. GFP-PRD-expressing cells treated with arsenite are indicated by arrow heads. (B) The percentage of GFP-positive cells containing SGs was determined and is shown in the graph. Error bars indicate s.e.m. (n = 3).
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pcbi.1004326.g002: TIA-1 is required for the assembly of SG.(A) GFP alone or GFP-PRD was transiently transfected into COS-7 cells as indicated. After 48 h, the cells were treated with arsenite (0.5 mM) for 50 min. Endogenous eIF4E, a SG marker protein, was visualized by immunofluorescence. GFP-PRD-expressing cells treated with arsenite are indicated by arrow heads. (B) The percentage of GFP-positive cells containing SGs was determined and is shown in the graph. Error bars indicate s.e.m. (n = 3).

Mentions: Next, the TIA-1 requirement for the assembly of SG was tested. TIA-1 possesses three RRMs at the N-terminus, along with a glutamine-rich PRD at the C-terminal region, both of which are essential for TIA-1-mediated SG assembly [9,29]. Previous studies reported that the expression of the C-terminal fragment of TIA-1, which contains the PRD alone, dominantly suppresses SG assembly [9,29]. Therefore, we constructed an expression vector encoding GFP-tagged PRD (GFP-PRD in S2B Fig), and examined if the expression of the PRD would affect the SG assembly. COS-7 cells were transiently transfected with either GFP alone (as control) or GFP-PRD, and incubated for 48 h. The cells were then treated with 0.5 mM arsenite for 50 min and the assembly of SGs was assessed by immunofluorescence microscopy (Fig 2A and 2B). In control cells expressing GFP alone, approximately 90 ± 76% of the cells formed SGs in response to arsenite. In contrast, cells expressing GFP-RPD scarcely showed SG assembly (10.4 ± 2.4%) (Fig 2B). These results clearly indicate that TIA-1 was required for the assembly of SG upon application of arsenite in COS-7 cells as was reported previously [8,29].


Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly.

Ohshima D, Arimoto-Matsuzaki K, Tomida T, Takekawa M, Ichikawa K - PLoS Comput. Biol. (2015)

TIA-1 is required for the assembly of SG.(A) GFP alone or GFP-PRD was transiently transfected into COS-7 cells as indicated. After 48 h, the cells were treated with arsenite (0.5 mM) for 50 min. Endogenous eIF4E, a SG marker protein, was visualized by immunofluorescence. GFP-PRD-expressing cells treated with arsenite are indicated by arrow heads. (B) The percentage of GFP-positive cells containing SGs was determined and is shown in the graph. Error bars indicate s.e.m. (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482703&req=5

pcbi.1004326.g002: TIA-1 is required for the assembly of SG.(A) GFP alone or GFP-PRD was transiently transfected into COS-7 cells as indicated. After 48 h, the cells were treated with arsenite (0.5 mM) for 50 min. Endogenous eIF4E, a SG marker protein, was visualized by immunofluorescence. GFP-PRD-expressing cells treated with arsenite are indicated by arrow heads. (B) The percentage of GFP-positive cells containing SGs was determined and is shown in the graph. Error bars indicate s.e.m. (n = 3).
Mentions: Next, the TIA-1 requirement for the assembly of SG was tested. TIA-1 possesses three RRMs at the N-terminus, along with a glutamine-rich PRD at the C-terminal region, both of which are essential for TIA-1-mediated SG assembly [9,29]. Previous studies reported that the expression of the C-terminal fragment of TIA-1, which contains the PRD alone, dominantly suppresses SG assembly [9,29]. Therefore, we constructed an expression vector encoding GFP-tagged PRD (GFP-PRD in S2B Fig), and examined if the expression of the PRD would affect the SG assembly. COS-7 cells were transiently transfected with either GFP alone (as control) or GFP-PRD, and incubated for 48 h. The cells were then treated with 0.5 mM arsenite for 50 min and the assembly of SGs was assessed by immunofluorescence microscopy (Fig 2A and 2B). In control cells expressing GFP alone, approximately 90 ± 76% of the cells formed SGs in response to arsenite. In contrast, cells expressing GFP-RPD scarcely showed SG assembly (10.4 ± 2.4%) (Fig 2B). These results clearly indicate that TIA-1 was required for the assembly of SG upon application of arsenite in COS-7 cells as was reported previously [8,29].

Bottom Line: SGs were assembled as a result of applying arsenite to HeLa cells.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Mathematical Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus