Limits...
Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry.

He L, Wu X, Simone J, Hewgill D, Lipsky PE - Nucleic Acids Res. (2005)

Bottom Line: Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells.Based upon this, TRAF2 homotrimerization could be detected.This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.

View Article: PubMed Central - PubMed

Affiliation: Flow Cytometry Section, Office of Science and Technology, National Institutes of Health Bethesda, MD 20892, USA. Lihe@mail.nih.gov

ABSTRACT
The availability of protein fluorophores with appropriate spectral properties has made it possible to employ fluorescence resonance energy transfer (FRET) to assess interactions between three proteins microscopically. Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells. Here, we report a flow-cytometric FRET technique that employed both direct energy transfer from CFP-->YFP-->mRFP and donor quenching to assess TRAF2 trimerization in living cells. Initially, a series of fusion proteins incorporating CFP, YFP and mRFP with spacers that did or did not permit FRET were employed to document the magnitude of CFP-->YFP and YFP-->mRFP FRET and to calculate the efficiency of CFP-->YFP-->mRFP two-step FRET. Based upon this, TRAF2 homotrimerization could be detected. This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.

Show MeSH

Related in: MedlinePlus

Validation of TRAF2 homotrimer formation in living cells by positive two-step FRET. (A) Flow cytometric profiles showing simultaneous measurements of FRET1 (CFP→YFP), FRET2 (YFP→mRFP) and two-step-FRET (CFP→YFP→mRFP) or potential FRET3 (CFP→mRFP). Region 3 (R3) with equal mRFP intensity for all transfected cells and region 4 (R4) were gated as positively and negatively transfected cells, respectively. The positive FRET1, FRET2 and two-step-FRET signals were detected in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4). In contrast, only FRET2 was detected in the cells co-expressing CFP/YFP-TRAF2/mRFP-TRAF2 (1), whereas only FRET1 was detected in the cells co-expressing CFP-TRAF2/YFP-TRAF2/mRFP (2). Weak positive FRET3 was detected in cells co-expressing CFP-TRAF2, YFP and mRFP-TRAF2 (3), indicating potential energy transfer from CFP-TRAF2 to mRFP-TRAF2. However, this transfer was significantly less than that detected in sample 4 (MFI: 32.7 versus 58.6). The MFIs of FRET1, FRET2 and two-step-FRET or potential FRET3 are shown for each FRET panel. (B) Flow cytometric histogram profiles showing the CFP→YFP FRET1 quenching and strong two-step-FRET signals, indicating trimerization of TRAF2 in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1074310&req=5

fig6: Validation of TRAF2 homotrimer formation in living cells by positive two-step FRET. (A) Flow cytometric profiles showing simultaneous measurements of FRET1 (CFP→YFP), FRET2 (YFP→mRFP) and two-step-FRET (CFP→YFP→mRFP) or potential FRET3 (CFP→mRFP). Region 3 (R3) with equal mRFP intensity for all transfected cells and region 4 (R4) were gated as positively and negatively transfected cells, respectively. The positive FRET1, FRET2 and two-step-FRET signals were detected in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4). In contrast, only FRET2 was detected in the cells co-expressing CFP/YFP-TRAF2/mRFP-TRAF2 (1), whereas only FRET1 was detected in the cells co-expressing CFP-TRAF2/YFP-TRAF2/mRFP (2). Weak positive FRET3 was detected in cells co-expressing CFP-TRAF2, YFP and mRFP-TRAF2 (3), indicating potential energy transfer from CFP-TRAF2 to mRFP-TRAF2. However, this transfer was significantly less than that detected in sample 4 (MFI: 32.7 versus 58.6). The MFIs of FRET1, FRET2 and two-step-FRET or potential FRET3 are shown for each FRET panel. (B) Flow cytometric histogram profiles showing the CFP→YFP FRET1 quenching and strong two-step-FRET signals, indicating trimerization of TRAF2 in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4).

Mentions: The next experiments examined whether this system could be used to assess interactions between three proteins in living cells. TRAF2 were chosen since it is known to form homotrimers in solution (22). As can be seen in Figure 6, FRET2 (YFP→mRFP), but not FRET1 (CFP→YFP), was detected in cells co-transfected with CFP, YFP-TRAF2 and mRFP-TRAF2 (1), and FRET1 (CFP→YFP) but not FRET2 (YFP→mRFP) was detected in those co-transfected with CFP-TRAF2, YFP-TRAF2 and mRFP (2). As a control, cells co-transfected with CFP-TRAF2, YFP and mRFP-TRAF2 (3) manifested a modest FRET3 (CFP→mRFP) signal (MFI of 32.7 in 3 versus 17.0 in 2 and 14.9 in 1). However, two-step FRET was significantly greater (MFI: 58.6) in cells co-transfected with CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4) whereas both FRET1 and FRET2 were positive (Figure 6B). FRET1 quenching was again observed in these cells (3.9 in 4 and 10.9 in 2, Figure 6B). These data are consistent with the formation of TRAF2 homotrimers.


Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry.

He L, Wu X, Simone J, Hewgill D, Lipsky PE - Nucleic Acids Res. (2005)

Validation of TRAF2 homotrimer formation in living cells by positive two-step FRET. (A) Flow cytometric profiles showing simultaneous measurements of FRET1 (CFP→YFP), FRET2 (YFP→mRFP) and two-step-FRET (CFP→YFP→mRFP) or potential FRET3 (CFP→mRFP). Region 3 (R3) with equal mRFP intensity for all transfected cells and region 4 (R4) were gated as positively and negatively transfected cells, respectively. The positive FRET1, FRET2 and two-step-FRET signals were detected in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4). In contrast, only FRET2 was detected in the cells co-expressing CFP/YFP-TRAF2/mRFP-TRAF2 (1), whereas only FRET1 was detected in the cells co-expressing CFP-TRAF2/YFP-TRAF2/mRFP (2). Weak positive FRET3 was detected in cells co-expressing CFP-TRAF2, YFP and mRFP-TRAF2 (3), indicating potential energy transfer from CFP-TRAF2 to mRFP-TRAF2. However, this transfer was significantly less than that detected in sample 4 (MFI: 32.7 versus 58.6). The MFIs of FRET1, FRET2 and two-step-FRET or potential FRET3 are shown for each FRET panel. (B) Flow cytometric histogram profiles showing the CFP→YFP FRET1 quenching and strong two-step-FRET signals, indicating trimerization of TRAF2 in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074310&req=5

fig6: Validation of TRAF2 homotrimer formation in living cells by positive two-step FRET. (A) Flow cytometric profiles showing simultaneous measurements of FRET1 (CFP→YFP), FRET2 (YFP→mRFP) and two-step-FRET (CFP→YFP→mRFP) or potential FRET3 (CFP→mRFP). Region 3 (R3) with equal mRFP intensity for all transfected cells and region 4 (R4) were gated as positively and negatively transfected cells, respectively. The positive FRET1, FRET2 and two-step-FRET signals were detected in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4). In contrast, only FRET2 was detected in the cells co-expressing CFP/YFP-TRAF2/mRFP-TRAF2 (1), whereas only FRET1 was detected in the cells co-expressing CFP-TRAF2/YFP-TRAF2/mRFP (2). Weak positive FRET3 was detected in cells co-expressing CFP-TRAF2, YFP and mRFP-TRAF2 (3), indicating potential energy transfer from CFP-TRAF2 to mRFP-TRAF2. However, this transfer was significantly less than that detected in sample 4 (MFI: 32.7 versus 58.6). The MFIs of FRET1, FRET2 and two-step-FRET or potential FRET3 are shown for each FRET panel. (B) Flow cytometric histogram profiles showing the CFP→YFP FRET1 quenching and strong two-step-FRET signals, indicating trimerization of TRAF2 in cells co-expressing CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4).
Mentions: The next experiments examined whether this system could be used to assess interactions between three proteins in living cells. TRAF2 were chosen since it is known to form homotrimers in solution (22). As can be seen in Figure 6, FRET2 (YFP→mRFP), but not FRET1 (CFP→YFP), was detected in cells co-transfected with CFP, YFP-TRAF2 and mRFP-TRAF2 (1), and FRET1 (CFP→YFP) but not FRET2 (YFP→mRFP) was detected in those co-transfected with CFP-TRAF2, YFP-TRAF2 and mRFP (2). As a control, cells co-transfected with CFP-TRAF2, YFP and mRFP-TRAF2 (3) manifested a modest FRET3 (CFP→mRFP) signal (MFI of 32.7 in 3 versus 17.0 in 2 and 14.9 in 1). However, two-step FRET was significantly greater (MFI: 58.6) in cells co-transfected with CFP-TRAF2, YFP-TRAF2 and mRFP-TRAF2 (4) whereas both FRET1 and FRET2 were positive (Figure 6B). FRET1 quenching was again observed in these cells (3.9 in 4 and 10.9 in 2, Figure 6B). These data are consistent with the formation of TRAF2 homotrimers.

Bottom Line: Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells.Based upon this, TRAF2 homotrimerization could be detected.This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.

View Article: PubMed Central - PubMed

Affiliation: Flow Cytometry Section, Office of Science and Technology, National Institutes of Health Bethesda, MD 20892, USA. Lihe@mail.nih.gov

ABSTRACT
The availability of protein fluorophores with appropriate spectral properties has made it possible to employ fluorescence resonance energy transfer (FRET) to assess interactions between three proteins microscopically. Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells. Here, we report a flow-cytometric FRET technique that employed both direct energy transfer from CFP-->YFP-->mRFP and donor quenching to assess TRAF2 trimerization in living cells. Initially, a series of fusion proteins incorporating CFP, YFP and mRFP with spacers that did or did not permit FRET were employed to document the magnitude of CFP-->YFP and YFP-->mRFP FRET and to calculate the efficiency of CFP-->YFP-->mRFP two-step FRET. Based upon this, TRAF2 homotrimerization could be detected. This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.

Show MeSH
Related in: MedlinePlus