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

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.

Figure 1. Lifetime variations of sEcGFP attached to OXPHOS subunits CoxVIIIa and CoxVIIc report specific molecular crowding at the contact site of CI₁CIII₂CIV₁.

(a) 3D maps of CII and the CI₁CIII₂CIV₁ supercomplex showing the side view (top) and the view of the supercomplex from the p-side (complex I in grey, complex III in darker grey and complex IV in light grey, cytochrome c in black). sEcGFP was fused to different subunits of respiratory complexes: CoxVIIc (yellow), CoxVIIIa (pink), CoxVIIIa-Link (violet), CoxIV (cyan), CIIC/SDHC (orange) and to a short matrix targeting sequence mt (green). Selected fitting models are shown. (b) Intensity lifetime image and time correlated single photon counting (TCSCP) histograms of mt-sEcGFP and CoxVIIIa-sEcGFP showing clear differences in lifetime revealed from the decay of fluorescence lifetime. Scale bars: 10 μm. (c) Lifetime constants τ for sEcGFP at different subunits of cytochrome c oxidase (CoxVIIIa - pink, CoxVIIc - yellow, and CoxIV - cyan), at subunit C of SDH (orange) and of matrix-targeted soluble sEcGFP (green). Each data point in the box-and-whisker plot represents the average fluorescence lifetime of a cell ( = one mitochondrial network). The error bars denote s.d., the boxes represent the 25^th to 75^th percentiles. The vertical lines in the boxes represent the median values, whereas the square symbols in the boxes denote the respective mean values. The minimum and maximum values are denoted by x. All measurements were performed at pH 7.1 to exclude pH effects. One data point per cell, error bars represent s.d. of ∼18 cells (n = 3 replicates). ***P < 0.001 (by one-way ANOVA).

Figure 2. 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 τ_amp 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).

Figure 3. Positioning of CoxIV-sEcGFP and CoxVIIIa-sEcGFP in a supercomplex and monomeric and dimeric CIV.

Models illustrating conformations expected for CoxVIIIa-sEcGFP in a supercomplex and in monomeric and homo-dimeric CIV₍₂₎ with respective environments: CoxVIIIa-sEcGFP senses a dense molecular environment in a supercomplex and an aqueous environment in free CIV₍₂₎ and CIV, while CoxIV-sEcGFP is always exposed to an aqueous environment. Supercomplex sensor CoxVIIIa-sEcGFP in pink framed with a dotted line, control CoxIV-sEcGFP in cyan, cytochrome c framed with disrupted lines.

Figure 4. FRET measurements showing close proximity of CIV subunit CoxVIIIa and CIII-subunit k in a supercomplex.

(a) Positions of CoxVIIIa-mRuby (acceptor, CIV-A) and CIIIk-Clover (donor, CIII-D) within a CICIII₂CIV supercomplex. Only one subunit k of one CIII is labeled. (b) Fluorescence lifetime of Clover as donor in the presence and absence of acceptor mRuby. Pair 1: CoxVIIIa-mRuby as acceptor and CIIIk-clover as donor. Pair 2: modified ATeam composed of clover as donor and mRuby as acceptor was used as FRET control. Significance: ***P < 0.001 compared to CIIIk-Clover (ANOVA one-way).