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Visualisation and identification of the interaction between STIM1s in resting cells.

He J, Yu T, Pan J, Li H - PLoS ONE (2012)

Bottom Line: Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s.Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB.Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.

View Article: PubMed Central - PubMed

Affiliation: Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. junhe@mails.tjmu.edu.cn

ABSTRACT
Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.

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FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.
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pone-0033377-g003: FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.

Mentions: In the current study, FRET measured by donor dequenching after acceptor photobleaching was also utilised to detect the interaction between STIM1s. HEK293T cells were cotransfected with eCFP-STIM1 and eYFP-STIM1 plasmids. After culture, laser scanning microscopy images were taken before and after photobleaching of the acceptor for 15 s with a 514 nm laser beam. Yellow fluorescence quenching and increase in the intensity of blue fluorescence were observed after the photobleaching. The transfer efficiency was calculated using the equation: E = 1–Fpre/Fpost. As shown in Fig. 3A and C, the average energy transfer efficiency for the cells withphotobleached acceptors in the resting state was 12.7% compared with only 1.7% in the controls. Following TG-induced Ca2+ store depletion, the energy transfer efficiency was 22.6% (Fig. 3 B, D).


Visualisation and identification of the interaction between STIM1s in resting cells.

He J, Yu T, Pan J, Li H - PLoS ONE (2012)

FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306384&req=5

pone-0033377-g003: FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.
Mentions: In the current study, FRET measured by donor dequenching after acceptor photobleaching was also utilised to detect the interaction between STIM1s. HEK293T cells were cotransfected with eCFP-STIM1 and eYFP-STIM1 plasmids. After culture, laser scanning microscopy images were taken before and after photobleaching of the acceptor for 15 s with a 514 nm laser beam. Yellow fluorescence quenching and increase in the intensity of blue fluorescence were observed after the photobleaching. The transfer efficiency was calculated using the equation: E = 1–Fpre/Fpost. As shown in Fig. 3A and C, the average energy transfer efficiency for the cells withphotobleached acceptors in the resting state was 12.7% compared with only 1.7% in the controls. Following TG-induced Ca2+ store depletion, the energy transfer efficiency was 22.6% (Fig. 3 B, D).

Bottom Line: Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s.Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB.Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.

View Article: PubMed Central - PubMed

Affiliation: Division of Histology and Embryology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. junhe@mails.tjmu.edu.cn

ABSTRACT
Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.

Show MeSH
Related in: MedlinePlus