Live-cell single-molecule labeling and analysis of myosin motors with quantum dots

Figures

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  FIGURE 1:. Approach for labeling intracellular proteins with QDs. (A) Schema for labeling intracellular proteins with QDs. (B) Binding of HaloTag ligand–QD with HaloTag proteins in vitro. HaloTag ligand–QDs were incubated with the lysate of KRX cells expressing HaloTag–14-3-3β/α proteins and separated on an agarose gel. Samples on the right half of the gel were denatured by boiling the lysates before mixing with HaloTag ligand–QDs. “Original” refers to QDs with no conjugation reaction. (C) Snapshots of QD fluorescence in 3T3-L1 fibroblasts. Cells were immersed with QD-containing solution, and electroporation was performed with the indicated poration pulse voltages. Solid and dashed lines represent plasma and nuclear membranes, respectively. The Laplacian of Gaussian-filtered images. (D) Dependence of internalization of QD in 3T3-L1 fibroblasts on poration pulse voltage.

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  FIGURE 2:. Tracking intracellular movement of HaloTag-myosin Vb. (A) QD fluorescence in a cell expressing HaloTag–myosin Vb labeled with HaloTag ligand–QDs. The Laplacian of Gaussian-filtered images. The solid line represents the plasma membrane. (B) Changes in the position of a labeled HaloTag–myosin Vb in the boxed region of A (arrowheads). (C) Trajectory of the particle shown in B. Color represents time as shown below the image. (D, E) MSD curve derived from the whole trajectory (D) and instantaneous diffusion coefficient (E) of the particle shown in B. Red solid and dashed lines in D are the best-fit curves with Eq. 2 and the linear component, respectively. The parameters are D = 0.028 μm2/s and v = 0.32 μm/s. The blue solid line in the inset of D is the best-fit curve obtained using Eq. 4 with a value of 1.8 for exponent α, indicating that the movement was mainly dominated by directional movement. (F) Mean MSD curves of HaloTag ligand–QDs in cells expressing HaloTag–myosin Vb (black) or no HaloTag proteins (gray). The red solid and dashed lines are the best-fit curves obtained using Eq. 2 and the linear component, respectively. The parameters are D = 0.007 μm2/s and v = 0.037 μm/s. The…

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  FIGURE 3:. Simultaneous observation of HaloTag–myosin Vb and F-actin. (A) Lifeact-Venus fluorescence. Trajectories of three HaloTag–myosin Vb labeled particles with HaloTag ligand–QDs are overlaid. Colors represent time as shown below the image. (B) Changes in the position of a HaloTag ligand–QD signal (magenta) along Lifeact-Venus–labeled F-actin (green) in the boxed region (i) in A. The Laplacian of Gaussian-filtered QD images is shown. (C) Magnified images overlaid with trajectories (a), MSD curves (b), instantaneous diffusion coefficients (c), and Lifeact-Venus intensities (d) of the three particles shown in A. The solid and dashed lines in (b) are the best-fit curves and linear components obtained using Eq. 2, respectively. The fitted parameters of particles i–iii are, respectively, D = 0.029, 0.071, and 0.11 and μm2/s and v = 0.40, 0.39, and 0.16 μm/s. Shaded areas in c and d represent QDs colocalized with Lifeact-Venus signals, which were estimated by the fluorescence intensity of Lifeact-Venus along the trajectories. Asterisks in c represent restricted movement of QDs on thick F-actin.

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  FIGURE 4:. Analysis of fast-moving HaloTag–myosin molecules. (A, B) Histograms of diffusion coefficients of HaloTag ligand–QDs calculated from whole trajectories in cells expressing no HaloTag proteins (A) or HaloTag–myosin Vb (B). The dashed line represents the set threshold (diffusion coefficient of mean +3 SDs in A), and the red bars in B represent particles exceeding the threshold. Data were calculated from 157 particles in three cells in A and 891 particles in nine cells in B. In B, 48 of 891 particles (5.4%) exceeded the threshold. (C) Trajectories of HaloTag ligand–QD signals above (left) and below (right) the threshold. Overlaid trajectories obtained from nine cells. (D) MSD curves calculated from particles above (red) and below (black) the threshold. An MSD curve from cells expressing no HaloTag protein (gray) is shown for comparison. Right, magnified graph within τ < 2 s and MSD < 0.06 μm2. The MSD curve of particles exceeding and below the threshold was best-fitted with Eq. 2 with parameters of D = 0.072 and 0.004 μm2/s and v = 0.14 and 0.01 μm/s, respectively (blue and green solid lines). Data are presented as mean ± SEM.

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  FIGURE 5:. Effect of insulin on HaloTag–myosin movement. (A) Lifeact-Venus fluorescence before (left) and after 4 min of insulin stimulation (right) in 3T3-L1 fibroblasts. Membrane ruffling (arrows) and actin comet tails (arrowhead) were observed. Images are maximum intensity projections over five successive frames. (B) Magnified images of boxed regions shown in A. (C) MSD curves derived from the whole part of the trajectories having diffusion coefficients >0.027 μm2/s in cells expressing HaloTag-myosin Ic (left), HaloTag-myh9 (center), and HaloTag-myosin Vb (right) before (black) and 5 min after (red) insulin stimulation. Data are presented as mean ± SEM.