Journal: The Journal of Biological Chemistry

Article Title: Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments * ^♦

doi: 10.1074/jbc.M114.609743

Figure Lengend Snippet: Complexity of thin filament activation. a, kymograph in sub-maximal activation conditions ( p Ca 6, 15 n m myosin, 0.1 μ m ATP) shows a continuous patch of activation that moves both toward the plus and minus ends of actin. The lower kymograph has the peak intensities labeled (using ImageJ skeletonize); this allow a clearer view of how these active regions collide and collapse catastrophically. The skeletonization was used only for visual purposes and not for the fitting used in b . This image is the first real time single molecule view of how the thin filament both activates and deactivates. b, center of a number of active regions was analyzed by measuring their positional displacement over a single frame and plotted as a histogram. This histogram follows a Gaussian distribution indicative of diffusion; the mean position (−0.88 ± 1.11 (S.D.) px) suggests the diffusion is unbiased. One pixel = 80 nm. c, global fitting of the intensity histogram data ( , b , d, and f ) across a number of conditions. The data in blue diamonds (10 fps) was obtained at a faster frame rate than those in red squares (3.8 fps) and were therefore fit separately; the results in either condition provide the same value for d of 11. The quality of the fits both validate the choice of model and provide an activation distance for the open state of 11 myosin-binding sites on actin. The remaining parameters used in this model are provided in . d, sensitivity was tested for each the parameters stated in used in the fitting for c . The value for each parameter was first halved, and the resulting ΔS.D. of the fit was squared and then summed with the ΔS.D. obtained when the same parameter was doubled. This root of this value is plotted as the root mean square ΔS.D. value (or sensitivity) for each parameter. A more sensitive value indicates the parameter is fit with better precision.

Article Snippet: To calculate the number of myosins bound for all clusters on individual thin filaments, we analyzed kymographic data directly by taking consecutive vertical slices through the kymograph and fitting each one with a custom-written (Matlab) multiple Gaussian fitting routine ( a ).

Techniques: Activation Assay, Labeling, Diffusion-based Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments * ^♦

doi: 10.1074/jbc.M114.609743

Figure Lengend Snippet: Analyzing the interactions between myosin and actin. a, vertical box is drawn one pixel wide and scanned along the time axis. Each box contains all of the fluorescence intensity information for a single time point in the movie along the length of a thin filament. In this example, one vertical slice is rotated 90° and plotted as a graph of intensity versus position along the thin filament ( lower graph in a ). This graph shows two peaks with a full width half-maximum of ∼300 nm (>100 actin monomers) as would be expected for a point source. The intensity of the peaks provides information on the number of myosins bound. Therefore, each vertical slice is fitted to a sum of Gaussian distributions with unconstrained intensities (shown as the fit line). The non-zero baseline represents the background noise, which is removed as a consequence of the fitting, which was performed using a custom-written Matlab routine. b, peak intensity values from all of the fitted Gaussian distributions were then plotted as a histogram ( blue squares ), and in this case the conditions are 15 n m myosin at p Ca 5 with 0.5 μ m ATP. As a result of this treatment, the histogram now represents steady-state intensities and has no spatial and temporal information from the kymograph. At low myosin concentrations without regulatory proteins, only single myosins were seen to bind actin resulting in a single peak for the intensity histogram (data not shown). This peak corresponds to the profile of a single eGFP. The next stage of analysis was to determine the number of intensity subpopulations that constitute a multiple myosin intensity histogram. This was achieved by fitting the histogram to multiple Gaussians, each with the standard deviation of a single eGFP until the fit could no longer be improved.

Article Snippet: To calculate the number of myosins bound for all clusters on individual thin filaments, we analyzed kymographic data directly by taking consecutive vertical slices through the kymograph and fitting each one with a custom-written (Matlab) multiple Gaussian fitting routine ( a ).

Techniques: Fluorescence, Standard Deviation

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Magnesium-induced folding of SL ribozyme constructs. Peak E shifts from the Gaussian fits of the FRET histograms at Mg2+ concentrations from 0.01 mM to 1 M for the SL (▪) and WT (⋄) 2WJ, 3WJ, and 4WJ ribozymes (parts A, B, and C, respectively). Peak E values for a control donor-acceptor labeled DNA molecule with dyes separated by 14 base pairs are shown for comparison as solid triangles in the 3WJ graph in part B.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Control, Labeling, Comparison

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Ratiometric spFRET data for the WT 4WJ ribozyme. (A) Example of donor (black) and acceptor (gray) emission bursts observed in spFRET experiments. A time trace is presented for the WT 4WJ ribozyme at a concentration of 100 pM in 50 mM Tris/HCl buffer with 0.1 mM Mg2+ at room temperature, and the data integration time per point was 0.5 ms. Fluorescent donor and acceptor bursts are observed above background. A buffer (background) time trace is also shown for comparison. Only bursts above a threshold of 40 counts (sum of donor and acceptor signals) were used to construct FRET efficiency histograms. (B) FRET efficiency histogram derived from calculation of FRET efficiencies from each accepted donor/acceptor event above a threshold of 40 counts, showing the docked and undocked subpopulations (see text), as well as the zero-E peak. The solid lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Concentration Assay, Comparison, Construct, Derivative Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: FRET efficiency histogram for a G11I mutant of the 4WJ ribozyme at 0.1 mM Mg2+, using the same conditions as for data presented in Fig. 3. Solid lines show fits using Gaussian functions. The relative area of the high efficiency (docked) peak is markedly reduced relative to the wild-type 4WJ ribozyme (Fig. 3), reflecting a destabilized tertiary structure for the mutant.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Mutagenesis

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Schematic representation of WT ribozyme constructs used for spFRET studies (upper panel). FRET efficiency histograms (bars) for wild-type 2WJ, 3WJ, and 4WJ hairpin ribozymes, showing undocked and docked ribozyme subpopulations at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Single-loop 2WJ, 3WJ, and 4WJ ribozyme constructs (upper panel) and corresponding FRET efficiency histograms at Mg2+ concentrations of 0.01, 0.1, 1, and 10 mM (lower panel). Data were acquired using a ribozyme concentration of 100 pM in 50 mM Tris/HCl buffer with Mg2+ at room temperature, and the data integration time per point was 0.5 ms. The smooth lines show fits using Gaussian functions.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques: Construct, Concentration Assay

Journal:

Article Title: Freely Diffusing Single Hairpin Ribozymes Provide Insights into the Role of Secondary Structure and Partially Folded States in RNA Folding

doi: 10.1529/biophysj.103.036087

Figure Lengend Snippet: Kinetic information from peak shape analysis of spFRET histograms. (A) Overlay of FRET efficiency histograms for SL (bold solid line) and WT 4WJ (data as bar graph, Gaussian fit as solid line) ribozymes at 10 mM Mg2+, emphasizing the asymmetric peak shape for the SL 4WJ. The error bars represent single standard deviations. (B) Overlay of FRET histograms from two-state simulations (lines) and experimental data (bars; errors represent single standard deviations) for the SL 4WJ at 10 mM Mg2+. The extended-undocked (EU)–quasi-docked (QD) equilibrium scheme is shown in the inset. Simulated histograms are shown for forward/reverse (k1/k−1) rate constants of 20 × 103/6.8 × 103 s−1 (bold solid line, 1), 41 × 103/14 × 103 s−1 (bold dashed line, 2), and 10 × 103/3.4 × 103 s−1 (bold dash-dot-dashed line, 3) with a corresponding equilibrium constant of 3 in favor of the quasi-docked state in all cases. See text for details.

Article Snippet: FRET histograms were fit with multiple Gaussian functions using Origin (OriginLab Corp., Northampton, MA), and the peak position ( E peak ) and relative areas of individual peaks were calculated from the fitting parameters.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Reconstitution of Proapoptotic BAK Function in Liposomes Reveals a Dual Role for Mitochondrial Lipids in the BAK-driven Membrane Permeabilization Process *

doi: 10.1074/jbc.M110.165852

Figure Lengend Snippet: Electrophysiological analysis of BAKΔC and tBID-mediated effect on planar BLMs. A–D, shown are representative current traces (shown as conductance G = I/V), and all point histograms corresponding to all tracings were obtained from at least three independent recordings at the conditions illustrated (left panels). The peak at ≈0 picosiemens (pS) represents the base-line conductance of the untreated BLMs. Black lines show Gaussian multi-pick fit (Origin software, Microcal); p > 0.99 for all fits.

Article Snippet: Black lines show Gaussian multi-pick fit (Origin software, Microcal); p > 0.99 for all fits.

Techniques: Software

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: PAS-mutant hERGs are sorted for lysosomal delivery from the cell surface. ( a ) hERG is targeted to LAMP1-positive endo-lysosomal compartments. Endocytic WT, M124R and C64Y hERG pool labelled by Ab capture (15 min at 37 °C) and remaining cell-surface hERG blocked with unconjugated secondary F(ab′) 2 (1 h on ice). Cells then chased at 37 °C for 3 h prior to fixation. Lysosomal compartments labelled with LAMP1 pAb. hERG (green) and LAMP1 (magenta) staining visualized by LCFM. Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 5 µm, right) images shown. Magnified area indicated by white box. Analysis of additional mutants (F29L, R56Q, T65P) in Supplementary Fig. . ( b ) Representative distribution of vesicular pH for WT and T65P hERG containing endocytic vesicles following 3 h chase. Overlay of multi-Gaussian peak-fits shown and mean pH ± SD indicated. N indicates total number of vesicles analyzed in a representative experiment. ( c ) PAS-mutations accelerate hERG endo-lysosomal delivery kinetics. Mean luminal pH of vesicles containing WT or T65P hERG measured by FRIA. Anti-HA Ab and FITC-Fab were bound on ice and FRIA was performed after 1- to 6-h chase. ( d ) Mean luminal pH of vesicles containing WT and PAS-mutant hERG following 3 h chase. ( e , f ) Lysosomal activity contributes to degradation of mature hERG proteins. Metabolic stability of WT and PAS-mutants hERG evaluated by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). V-ATPase inhibition with Bafilomycin A1 (BafA1, 200 nM), or proteasome inhibition with Bortezomib (Bort, 3 µM) or Ixazomib (Ixa, 3 µM) attenuated the rapid degradation of PAS-mutants. Mature complex-glycosylated (~155 kDa) and ER-resident core-glycosylated (~135 kDa) hERG indicated by solid and empty arrows, respectively. Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See methods and materials for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Mutagenesis, Staining, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: Peripheral quality control engagement is dependent on conformational destabilization. ( a ) Mature hERG is destabilized at elevated temperature. Metabolic stability of mature WT, PAS-mutant (F29L and T65P) or temperature-rescued G601S (48 h at 26 °C, rG601S) hERG evaluated at 37 °C or 41 °C by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. ( b ) Turnover kinetics of mature WT and F29L hERG fit using single-exponential decay functions. Similar results obtained for T65P and rG601S hERG (Supplementary Fig. ). ( c ) Turnover rate-constants determined by curve fitting as in ( b ) and expressed as fold increase relative to 37 °C. ( d ) Pharmacological correction of hERG folding restores cell-surface stability. PM-turnover of WT and select PAS-mutants hERG measured by cell-surface ELISA following overnight (16 h) E4031 treatment (10 µM). ( e ) Pharmacochaperone treatment improves folding of nascent hERG at the ER but does not promote refolding of mature channels at the PM. Internalization of WT and select PAS-mutant hERG measured by PM-ELISA following acute (1 h) or overnight (16 h) E4031 pre-treatment (10 µM). ( f ) Delivery of PM-labelled T65P hERG to LAMP1-positive compartments evaluated by LCFM following 3 h chase. Lysosomal delivery is prevented by overnight pre-treatment with E4031 (10 µM). Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 2 µm, right) images shown. Magnified area indicated by white box. Analysis of WT and additional PAS-mutants in Supplementary Fig. . ( g ) Pharmacochaperone pre-treatment prevents endo-lysosomal trafficking of T65P hERG. Representative histogram of T65P hERG vesicular pH following 3 h chase. Overlay of multi-Gaussian peak-fits (mean ± SD) shown. N indicates total number of vesicles evaluated. ( h ) Mean luminal pH of hERG-containing endocytic vesicles measured by FRIA following overnight treatment with E4031 (10 µM) and 3 h chase at 37 °C. ( i ) Subset of temperature-rescued PAS-mutants are resistant to unfolding at physiological temperature. Internalization of WT and PAS-mutant hERG measured by PM-ELISA following low-temperature rescue (30 °C for 24 h) and unfolding (37 °C for 2 h). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Control, Mutagenesis, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay