Z.W. Zhao, J.C.M. Gebhardt, D,M. Suter and X.S. Xie
Reply to "Convergence of chromatin binding estimates in live cells"
Nat. methods 10 (2013) 692
A.A. Torrano, J. Blechinger, C. Osseforth, C. Argyo, A. Reller, T. Bein, J. Michaelis and C. Bräuchle
A fast analysis method to quantify nanoparticle uptake on a single cell level
This study examines the absolute quantification of particle uptake into cells. Methods: We developed a novel method to analyze stacks of confocal fluorescence images of single cells interacting with nanoand micro-particles. Particle_in_Cell-3D is a freely available ImageJ macro. During the image analysis routine, single cells are reconstructed in 3D and split into two volumes - intracellular and the membrane region. Next, particles are localized and color-coded accordingly. The mean intensity of single particles, measured in calibration experiments, is used to determine the absolute number of particles. Results: Particle_in_Cell-3D was successfully applied to measure the uptake of 80-nm mesoporous silica nanoparticles into HeLa cells. Furthermore, it was used to quantify the absolute number of 100-nm polystyrene nanoparticles forming agglomerates of up to five particles; the accuracy of these results was confirmed by super-resolution, stimulated emission depletion microscopy. Conclusion: Particle_in_Cell-3D is a fast and accurate method that allows the quantification of particle uptake into cells.
J.C.M. Gebhardt*, D.M. Suter*, R. Roy, Z. Zhao, A.R. Chapman, S. Basu, T. Maniatis and X.S. Xie
Single-molecule imaging of transcription factor binding to DNA in live mammalian cells
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.
Nat. Methods 10 (2013) 421-42
M. Hinczewski, J.C.M. Gebhardt, M. Rief, and D. Thirumalai
From mechanical folding trajectories to intrinsic energy landscapes of proteins
In single-molecule laser optical tweezer (LOT) pulling experiments, a protein or RNA is juxtaposed between DNA handles that are attached to beads in optical traps. The LOT generates folding trajectories under force in terms of time-dependent changes in the distance between the beads. How to construct the full intrinsic folding landscape (without the handles and beads) from the measured time series is a major unsolved problem. By using rigorous theoretical methods - which account for fluctuations of the DNA handles, rotation of the optical beads, variations in applied tension due to finite trap stiffness, as well as environmental noise and limited bandwidth of the apparatus - we provide a trac-table method to derive intrinsic free-energy profiles. We validate the method by showing that the exactly calculable intrinsic free-energy profile for a generalized Rouse model, which mimics the two-state behavior in nucleic acid hairpins, can be accurately extracted from simulated time series in a LOT setup regardless of the stiffness of the handles. We next apply the approach to trajectories from coarse-grained LOT molecular simulations of a coiled-coil protein based on the GCN4 leucine zipper and obtain a free-energy landscape that is in quantitative agreement with simulations performed without the beads and handles. Finally, we extract the intrinsic free-energy landscape from experimental LOT measurements for the leucine zipper.
Proc. Natl. Acad. Sci. 110 (2013) 4500-4505