3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria

@inproceedings{Renz20153DlocalizationMA,
  title={3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria},
  author={Anja Renz and Marc Renz and Diana Kl{\"u}tsch and Gabriele Deckers-Hebestreit and Michael B{\"o}rsch},
  booktitle={Photonics West - Biomedical Optics},
  year={2015}
}
FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of… 
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References

SHOWING 1-10 OF 117 REFERENCES
Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy
FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP).
Microscopy of single FoF1‐ATP synthases— The unraveling of motors, gears, and controls
TLDR
Förster resonance energy transfer, which has been used for simultaneous monitoring of conformational changes of different parts of this rotary motor, is one of them and may become the tool for the analysis of single FoF1‐ATP synthases in membranes of living cells.
Imaging and quantification of trans-membrane protein diffusion in living bacteria.
TLDR
A novel approach, IPODD (inverse projection of displacement distributions), is introduced to obtain diffusion coefficients from the usually obtained 2-D projected diffusion trajectories of the highly 3-D curved bacterial membrane to provide correct diffusion coefficients and allow unravelling of heterogeneously diffusing populations.
The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap
TLDR
This work labels F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer and compares FRET changes in single F1 and FRET histograms for different biochemical conditions to evaluate the proposed regulatory mechanism.
How to quantify protein diffusion in the bacterial membrane
TLDR
A method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs), and demonstrates the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins.
Spotlighting motors and controls of single FoF1-ATP synthase.
TLDR
The present mini-review summarizes smFRET rotation experiments and previews newsmFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.
Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET
TLDR
Progress is reported of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.
Monitoring the rotary motors of single FoF1-ATP synthase by synchronized multi channel TCSPC
TLDR
The action mode of bactericidal drugs, i.e. inhibitors of FoF1-ATP synthase like aurovertin, could be investigated by the time resolved single-molecule FRET approach.
3D-localization of the a-subunit in F0F1-ATP synthase by time resolved single-molecule FRET
TLDR
Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps and the stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes and the position of the FRET donors at the asubunit was calculated.
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