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Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells

View Article: PubMed Central - PubMed

ABSTRACT

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

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The lifetime of CoxVIIIa-sEcGFP increases in cells with decreased levels of scaffold proteins.(a) Silencing of HIGD2A and CoxVIIa2l in stable CoxVIIIa-sEcGFP cells as shown by immuno-staining. Scrambled siRNA was used as a control, loading control was VDAC (middle panel). Right panel: ratio between HIGD2A respectively CoxVIIa2l and VDAC levels. (b) TCSPC diagram showing the change in fluorescence decay with decreased scaffold proteins. (c) Average fluorescence lifetimes τamp of stable HeLa cell lines expressing CoxVIIIa-sEcGFP, CoxIV-sEcGFP and mt-sEcGFP, respectively with downregulated HIGD2A or CoxVIIa2l. One data point per cell, error bars represent s.d. of ∼24 cells (n = 3 biological replicates). (d) Fluorescence intensity/lifetime images of CoxVIIIa-sEcGFP in cells with decreased scaffold protein HIGD2A or All-Stars non-targeting siRNA. Scale bars: 10 μm. Significance: ***P < 0.001 compared to CoxVIIIa-sEcGFP (ANOVA one-way).
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f2: The lifetime of CoxVIIIa-sEcGFP increases in cells with decreased levels of scaffold proteins.(a) Silencing of HIGD2A and CoxVIIa2l in stable CoxVIIIa-sEcGFP cells as shown by immuno-staining. Scrambled siRNA was used as a control, loading control was VDAC (middle panel). Right panel: ratio between HIGD2A respectively CoxVIIa2l and VDAC levels. (b) TCSPC diagram showing the change in fluorescence decay with decreased scaffold proteins. (c) Average fluorescence lifetimes τamp of stable HeLa cell lines expressing CoxVIIIa-sEcGFP, CoxIV-sEcGFP and mt-sEcGFP, respectively with downregulated HIGD2A or CoxVIIa2l. One data point per cell, error bars represent s.d. of ∼24 cells (n = 3 biological replicates). (d) Fluorescence intensity/lifetime images of CoxVIIIa-sEcGFP in cells with decreased scaffold protein HIGD2A or All-Stars non-targeting siRNA. Scale bars: 10 μm. Significance: ***P < 0.001 compared to CoxVIIIa-sEcGFP (ANOVA one-way).

Mentions: To verify that the decreased lifetime of sEcGFP at CoxVIIIa was an effect of defined supercomplex assembly but not caused by random molecular crowding or by the short distance of the probe to the membrane surface, we determined the lifetime of sEcGFP attached to subunit C of complex II. OXPHOS complex CII is a monomeric protein complex16 found in cristae as CI, CIII and CIV31. sEcGFP was fused to the C-terminus of the C-subunit of CII (SDHC) with a 7 amino acid residues linker that should result in a short distance between the probe and the membrane surface (Table 1). The resulting fluorescence lifetime τ = 2.15 ns ( ± 0.05 ns, s.d.) for CIIC-sEcGFP was similar to the lifetime of CoxIV- sEcGFP (τ = 2.12 ns ± 0.03 ns, s.d.) at the surface of CIV. It was about 0.47 ns higher than that for CoxVIIc-sEcGFP with the same linker length between the membrane domain and the sEcGFP (Table 1). Overall, the fluorescence lifetimes of CoxVIIc-sEcGFP and CoxVIIIa-sEcGFP were the shortest of all lifetimes measured by a significant margin, and this is consistent with a position in the center of a supercomplex (Fig. 2c). Similar results were obtained with mCitrine as sensor – except that the τ values were shifted to longer τ(Δ~0.59 ns) due to the generally longer lifetime of mCitrine (Supplementary Fig. S5)32.


Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells
The lifetime of CoxVIIIa-sEcGFP increases in cells with decreased levels of scaffold proteins.(a) Silencing of HIGD2A and CoxVIIa2l in stable CoxVIIIa-sEcGFP cells as shown by immuno-staining. Scrambled siRNA was used as a control, loading control was VDAC (middle panel). Right panel: ratio between HIGD2A respectively CoxVIIa2l and VDAC levels. (b) TCSPC diagram showing the change in fluorescence decay with decreased scaffold proteins. (c) Average fluorescence lifetimes τamp of stable HeLa cell lines expressing CoxVIIIa-sEcGFP, CoxIV-sEcGFP and mt-sEcGFP, respectively with downregulated HIGD2A or CoxVIIa2l. One data point per cell, error bars represent s.d. of ∼24 cells (n = 3 biological replicates). (d) Fluorescence intensity/lifetime images of CoxVIIIa-sEcGFP in cells with decreased scaffold protein HIGD2A or All-Stars non-targeting siRNA. Scale bars: 10 μm. Significance: ***P < 0.001 compared to CoxVIIIa-sEcGFP (ANOVA one-way).
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f2: The lifetime of CoxVIIIa-sEcGFP increases in cells with decreased levels of scaffold proteins.(a) Silencing of HIGD2A and CoxVIIa2l in stable CoxVIIIa-sEcGFP cells as shown by immuno-staining. Scrambled siRNA was used as a control, loading control was VDAC (middle panel). Right panel: ratio between HIGD2A respectively CoxVIIa2l and VDAC levels. (b) TCSPC diagram showing the change in fluorescence decay with decreased scaffold proteins. (c) Average fluorescence lifetimes τamp of stable HeLa cell lines expressing CoxVIIIa-sEcGFP, CoxIV-sEcGFP and mt-sEcGFP, respectively with downregulated HIGD2A or CoxVIIa2l. One data point per cell, error bars represent s.d. of ∼24 cells (n = 3 biological replicates). (d) Fluorescence intensity/lifetime images of CoxVIIIa-sEcGFP in cells with decreased scaffold protein HIGD2A or All-Stars non-targeting siRNA. Scale bars: 10 μm. Significance: ***P < 0.001 compared to CoxVIIIa-sEcGFP (ANOVA one-way).
Mentions: To verify that the decreased lifetime of sEcGFP at CoxVIIIa was an effect of defined supercomplex assembly but not caused by random molecular crowding or by the short distance of the probe to the membrane surface, we determined the lifetime of sEcGFP attached to subunit C of complex II. OXPHOS complex CII is a monomeric protein complex16 found in cristae as CI, CIII and CIV31. sEcGFP was fused to the C-terminus of the C-subunit of CII (SDHC) with a 7 amino acid residues linker that should result in a short distance between the probe and the membrane surface (Table 1). The resulting fluorescence lifetime τ = 2.15 ns ( ± 0.05 ns, s.d.) for CIIC-sEcGFP was similar to the lifetime of CoxIV- sEcGFP (τ = 2.12 ns ± 0.03 ns, s.d.) at the surface of CIV. It was about 0.47 ns higher than that for CoxVIIc-sEcGFP with the same linker length between the membrane domain and the sEcGFP (Table 1). Overall, the fluorescence lifetimes of CoxVIIc-sEcGFP and CoxVIIIa-sEcGFP were the shortest of all lifetimes measured by a significant margin, and this is consistent with a position in the center of a supercomplex (Fig. 2c). Similar results were obtained with mCitrine as sensor – except that the τ values were shifted to longer τ(Δ~0.59 ns) due to the generally longer lifetime of mCitrine (Supplementary Fig. S5)32.

View Article: PubMed Central - PubMed

ABSTRACT

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

No MeSH data available.


Related in: MedlinePlus