Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Soluble (non-MT) tubulin activates processive motility of HSET on single MTs. a Schematic. EGFP-HSET truncations were diluted in P12 buffer and monitored on GMPCPP-stabilized MTs by time-lapse TIRF. b Representative kymographs for time-lapse TIRF images for the indicated constructs at the following concentrations: EGFP-HSET and EGFP-HSETΔMotor, 50 pM. EGFP-HSETΔTail, 250 pM. Distance is on the x -axis (scale bar, 10 µm), and time is on the y -axis (scale bar, 10 s). c Mean-squared displacement (MSD) analysis of particle motion. The reported diffusion constant D is determined from a linear fit over the first 5 s, with the units nm 2 /s: EGFP-HSET: D = 6.3 × 10 4 , n = 206; EGFP-HSETΔMotor: D = 9.4 × 10 4 , n = 197; EGFP-HSETΔTail: D = 0.1 × 10 4 , n = 200. Data are presented as the calculated mean MSD ( y -axis) from two independent experiments over the indicated time intervals ( x -axis) for the indicated n particles ± SEM. d EGFP-HSET in BRB80 + 50 mM KCl was observed in the absence (left) or presence (right) of soluble tubulin and visualized by kymograph ( x -scale bar, distance, 10 µm; y -scale bar, time, 1 min). e Quantification of processive (≥5 s) event frequency as a function of [EGFP-HSET] in the presence (dark green) or absence (light green) of 2 µM tubulin. Data are presented as the number of processive events divided by the total observed MT length multiplied by the observation time for two independent experiments ± SD calculated from N ≥ 10 movies for each condition. Boxes represent first and third quartiles, whiskers represent detection limits, and lines represent median (mean overlaid). f Unlabeled HSET was mixed with 10 nM Cy5-tubulin in BRB80 + 50 mM KCl and observed. Velocities and run lengths of moving Cy5-tubulin particles were determined by kymograph and plotted as histograms. Data are reported as the mean velocity and run length values of n particles from CDF fitting ± the 95% CI from bootstrapping from two independent experiments. g 100 nM Cy5-tubulin (magenta) and 1 nM EGFP-HSET (green) were observed near-simultaneously by high-speed TIRF in BRB80 + 50 mM KCl, and visualized by kymograph ( x -scale bar, distance, 5 µm; y -scale bar, time, 10 s)

Article Snippet: Diffusion coefficients were calculated using the MSD analyzer MATLAB plugin , where the first 5 s were used for MSD analysis.

Techniques: Construct, Diffusion-based Assay

Journal: Methods in enzymology

Article Title: Direct Quantitative Monitoring of Homology-Directed DNA Repair of Damaged Telomeres

doi: 10.1016/bs.mie.2017.11.010

Figure Lengend Snippet: (A) Equation of mean square displacement (MSD) during Δt intervals for a particle i traveling in a 2D plane. (B) MSD plots of different classes of mobility. The equation, MSD= Γ tα, is used to provide a best fit curve for the MSD trajectories. Coefficient Γ indicates the magnitude of travel, while α is a time-dependence coefficient which indicates the type of movement the particle exhibits. (i) α Values of less than 1 indicate subdiffusion resulting from restriction to diffusion which would occur during molecular crowding in the nucleus. (ii) α Values of 1 indicate Brownian diffusion. (iii) α Values of greater than 1 indicate superdiffusive mobility. An α value of 2 would be characteristic of a particle traveling in a directed manner (Guigas & Weiss, 2008; Marshall et al., 1997; Zajac, Goldman, Holzbaur, & Ostap, 2013).

Article Snippet: DMEM cell culture medium with GlutaMAX (ThermoFisher), supplemented with 10% bovine calf serum and 1% Penicillin/Streptomycin (Gibco) 6-Well culture plates (Sarstedt) LipoD293 transfection reagent (Signagen) 22×22mm No 1.5 cover glass (Electron Microscopy Sciences) Incubator, 37°C and 5% CO 2 Leibovitz’s L-15 medium with l -glutamine, without phenol red (ThermoFisher) 4-Hydroxytamoxifen (Sigma) at 1m M stock concentration Shield-1 ligand (Clontech) at 0.5m M stock concentration Inverted fluorescence microscope (DM6000, Leica Microsystems), equipped with a charge-coupled device camera (QuantEM 512C, Photometrics), a 100×1.4 NA objective, automated XYZ stage (Ludl Electronic Products), an X-LIGHT Confocal Imager (Crisel Electrooptical Systems), and a SPECTRA X Light Engine (Lumencor) Stagetop heating incubator (Tokai Hit) Magnetic coverslip mounting chamber (Chamlide CM-S22–1, LCI) MetaMorph software (Molecular Devices) ImageJ Fiji (NIH, http://fiji.sc/ ) TrackMate plugin ( http://imagej.net/TrackMate ) StackReg plugin (Dr. Philippe Théevenaz, EPFL) MATLAB software (MathWorks) Mean square displacement (MSD) analyzer scripts (Dr. Jean-Yves Tinevez, Institut Pasteur) importTrackMateTracks ( https://github.com/fiji/TrackMate/blob/master/scripts/importTrackMateTracks.m ) MSD analyzer ( https://github.com/tinevez/msdanalyzer )

Techniques: Diffusion-based Assay