Limits...
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

Assembly of SGs by arsenite.(A) SGs were visualized by green fluorescent protein (GFP)-tagged TIA1 in living HeLa cells. Arsenite (0.5 mM) was applied to HeLa cells at 0 min, and SGs were seen as punctate structures after 10 min. SGs gradually localized at perinuclear region. (B) The number of SG increased after a short latency reaching a peak around 30 min, and then it decreased. Data shown is the average of 9 cells. (C) The increase in the average size of SG was sublinear. SG size was averaged for all SGs in 9 cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482703&req=5

pcbi.1004326.g001: Assembly of SGs by arsenite.(A) SGs were visualized by green fluorescent protein (GFP)-tagged TIA1 in living HeLa cells. Arsenite (0.5 mM) was applied to HeLa cells at 0 min, and SGs were seen as punctate structures after 10 min. SGs gradually localized at perinuclear region. (B) The number of SG increased after a short latency reaching a peak around 30 min, and then it decreased. Data shown is the average of 9 cells. (C) The increase in the average size of SG was sublinear. SG size was averaged for all SGs in 9 cells.

Mentions: First we investigated the assembly of SGs in HeLa cells. To monitor the process of SG assembly in living cells, HeLa cells were transiently transfected with an expression plasmid for green fluorescent protein (GFP)-tagged TIA-1, an SG-nucleating protein. 40 h after transfection, cells were stimulated with arsenite, and the time-lapse fluorescence images were acquired every 5 min for 55 min. SGs were clearly observed at 10 min (green dots in Fig 1A). The size of SGs was small at this stage, but grew larger at a later time (50 min). In contrast, the number of SGs reached a maximum at 30 min, then decreased as a function of time. These dynamics to exemplify SG assembly are qualitatively shown in Fig 1B and 1C. A small number of SG assembly was observed at 5 min reaching a maximum (31.7 ± 2.7, N = 9) at 30 min, and then decreased to 22.0 ± 2.8 at 55 min (Fig 1B). During these observable changes in the number of SGs, the average size increased monotonically with time (Fig 1C). In this quantification, the size of SGs were measured by the number of pixels (Materials and Methods). S1 Movie demonstrates the overall dynamics clearly and was similar to previous reports [37,39–41]. To validate our SG size measurement, we quantified SGs differently by integrated fluorescent intensity aimed at SG size measurement by volume instead of diameter (Materials and Methods). The time course of SG assembly and its evolution of average size were not changed significantly (left and middle panels in S2A Fig).


Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly.

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

Assembly of SGs by arsenite.(A) SGs were visualized by green fluorescent protein (GFP)-tagged TIA1 in living HeLa cells. Arsenite (0.5 mM) was applied to HeLa cells at 0 min, and SGs were seen as punctate structures after 10 min. SGs gradually localized at perinuclear region. (B) The number of SG increased after a short latency reaching a peak around 30 min, and then it decreased. Data shown is the average of 9 cells. (C) The increase in the average size of SG was sublinear. SG size was averaged for all SGs in 9 cells.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004326.g001: Assembly of SGs by arsenite.(A) SGs were visualized by green fluorescent protein (GFP)-tagged TIA1 in living HeLa cells. Arsenite (0.5 mM) was applied to HeLa cells at 0 min, and SGs were seen as punctate structures after 10 min. SGs gradually localized at perinuclear region. (B) The number of SG increased after a short latency reaching a peak around 30 min, and then it decreased. Data shown is the average of 9 cells. (C) The increase in the average size of SG was sublinear. SG size was averaged for all SGs in 9 cells.
Mentions: First we investigated the assembly of SGs in HeLa cells. To monitor the process of SG assembly in living cells, HeLa cells were transiently transfected with an expression plasmid for green fluorescent protein (GFP)-tagged TIA-1, an SG-nucleating protein. 40 h after transfection, cells were stimulated with arsenite, and the time-lapse fluorescence images were acquired every 5 min for 55 min. SGs were clearly observed at 10 min (green dots in Fig 1A). The size of SGs was small at this stage, but grew larger at a later time (50 min). In contrast, the number of SGs reached a maximum at 30 min, then decreased as a function of time. These dynamics to exemplify SG assembly are qualitatively shown in Fig 1B and 1C. A small number of SG assembly was observed at 5 min reaching a maximum (31.7 ± 2.7, N = 9) at 30 min, and then decreased to 22.0 ± 2.8 at 55 min (Fig 1B). During these observable changes in the number of SGs, the average size increased monotonically with time (Fig 1C). In this quantification, the size of SGs were measured by the number of pixels (Materials and Methods). S1 Movie demonstrates the overall dynamics clearly and was similar to previous reports [37,39–41]. To validate our SG size measurement, we quantified SGs differently by integrated fluorescent intensity aimed at SG size measurement by volume instead of diameter (Materials and Methods). The time course of SG assembly and its evolution of average size were not changed significantly (left and middle panels in S2A Fig).

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