Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation

Abstract

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.

Partition, orientation and mobility of ubiquinones in a lipid bilayer

“…Results from other methods such as fluorescence recovery after photo-bleaching probe rather different length scales (µm) and yield slower diffusion constants in the order of 10 −8 to 10 −9 cm 2 s −1 [17,21]. Thus, we conclude that results for the calculated diffusion constants are in the same order of magnitude to experimental measurements for both POPC pure lipid bilayers [76] and for ubiquinone embedded in the bilayer [18].…”

“…There is also a lack of consensus on the dynamical properties of ubiquinone embedded in lipid bilayers [17,18,19,20,21] as diffusion constants experimentally determined range from 10 −9 up to 10 −6 cm 2 s −1 . This high variation is due to the application of various measurement techniques [22,19] and to the usage of probes (fluorescent, spin labels, etc.)…”

Analytical tools for single-molecule fluorescence imaging in cellulo

“…This is important not simply on a qualitative level but rather the position of identified peaks in a distribution can be robustly quantified to identify distinct single-molecule states. As microscope camera sensitivity, the photophysical properties of new fluorophores, and the methods of delivering fluorophores inside cells with specificity improve, single-molecule fluorescence imaging experiments in live cells will become increasingly more ambitious in terms of the practical aspects of imaging single molecular complexes in living cells 70 ; truly multi-dimensional imaging using not just multiple colours 71 but multiple polarization states as well as simultaneous electrical and chemical measurements will most likely become increasingly more prevalent. Such complex datasets will ideally provide correlated and orthogonal information of molecular and cellular properties, necessitating yet further objective, analytical tools for extraction of molecular level information in a noisy environment.…”

“…Reliable detection of fluorescently-labelled single molecule also depends on their local density, namely the nearest neighbour separation distance; if this distance is comparable or less than the optical resolution limit as set by the PSF of the imaging system then there is a higher probability fluorophores will no longer be resolved individually as distinct molecules. The effect of increasing concentration of photoactive molecules on nearest-neighbour distance can be characterized analytically by using a Poisson nearest-neighbour model function 8,43 to generate maps for dependence of the likelihood of 'chance' colocalization (i.e. that two particles in a live cell will be separated randomly by less than the optical resolution limit) as a function of both the stoichiometry of diffusing particles (i.e.…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…Nanoscale tracking determined the position of tracked Mig1 foci to a lateral precision of 40 nm [32,62] coupled to stoichiometry analysis using stepwise photobleaching of GFP [52] and single cell copy number analysis [63]. An additional output from the tracking was the effective diffusion coefficient D as a function of its location in either the cytoplasm, nucleus or translocating across the nuclear envelope, as well as the copy number of Mig1 molecules associated with each subcellular region and in each cell as a whole, indicating ~850–1,300 Mig1 molecules per cell dependent on extracellular glucose.…”