Volume 18, Issue 20, Pages 1581-1586 (October 2008) Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles  Irina Semenova, Anton Burakov, Neda Berardone, Ilya Zaliapin, Boris Slepchenko, Tatyana Svitkina, Anna Kashina, Vladimir Rodionov  Current Biology  Volume 18, Issue 20, Pages 1581-1586 (October 2008) DOI: 10.1016/j.cub.2008.08.070 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Treatment of Melanophores with Actin Filament-Stabilizing Drugs Inhibits Transport of Pigment Granules (A) Phase-contrast images of melanophores treated with melatonin to induce pigment aggregation (top row) or with melanocyte-stimulating hormone to induce pigment dispersion (bottom row) in the absence (left panel) or in the presence (right panel) of jasplakinolide (1 μM). Pairs of images in each panel show the same cells before or 10 min after hormone treatment. Whereas pigment aggregation occurred with a normal kinetics, the rate of pigment dispersion was significantly inhibited in jasplakinolide-treated cells. Numbers indicate time in min; The scale bar represents 20 μm. (B) Quantification of actin-dependent movement of pigment granules measured in cells with disrupted cytoplasmic microtubules. Plots show changes with time of averaged squared distances traveled by pigment granules from the starting point in control cells (black squares), cells treated with jasplakinolilde (1 μM) for 5 min (red circles), cells injected with phalloidin solution (100 μM; purple triangles), buffer-injected cells (blue diamonds), GFP-overexpressing cells (green triangles), or cells overexpressing dominant-negative myosin Va construct (MST-GFP; dark blue triangles). Treatment of cells with jasplakinolide or microinjection with phalloidin solution reduces the movement of pigment granules to the levels seen in melanophores overexpressing dominant-negative myosin Va. Error bars indicate mean ± SD. (C) Decomposition of the trajectories of pigment-granule movement in cells with disrupted microtubules into linear (blue) and random diffusion-like displacements (green) through multiscale trend analysis in control (two top trajectories) and jasplakinolide-treated (two bottom trajectories) cells. The lengths of linear segments of the trajectories that correspond to actin-based runs are significantly shorter in jasplakinolide-treated cells. See also Movies S1–S6. Current Biology 2008 18, 1581-1586DOI: (10.1016/j.cub.2008.08.070) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Jasplakinolide Treatment Stabilizes Actin Filaments, but Does Not Significantly Change Their Organization, or the Levels of Actin Polymer in the Cytoplasm (A and B) FRAP analysis of the turnover rates of actin filaments in control and jasplakinolide-treated cells. (A) Sets of successive images of the bleached zones in control nontreated (left) or jasplakinolide-treated (right) cells. The scale bar represents 10 μm. (B) Quantification of fluorescence recovery in the bleached zones located at approximately equal distances from the cell center and cell margin in the areas of the cytoplasm capable of supporting actin-based transport. Black squares represent control cells; gray circles represent jasplakinolide-treated cells. Error bars represent SEM for measurements in ten different cells. Recovery of actin fluorescence is significantly faster in control than in jasplakinolide-treated cells. Numbers shown on (A) indicate time after the photobleaching. (C) Distribution of actin fluorescence in the same cell before (middle) or 5 min after (right) application of jasplakinolide. For the labeling of actin filaments, the cell was injected with rhodamine-actin 2 hr prior to acquisition of images. The left, phase-contrast image that shows distribution of pigment granules in the same cell; jasplakinolide treatment does not significantly change the distribution of actin fluorescence. The scale bar represents 20 μm. (D) Electron micrographs of platinum replicas of cytoplasmic regions located at approximately equal distances from the cell center and cell margin in control nontreated (left) and a jasplakinolide-treated (right) melanophores. The characteristic rope-like appearance of the actin filaments (inserts), which allowed their identification in electron micrographs, is explained by their decoration with the S1 subfragment of myosin during the preparation of samples for electron microscopy [7]. The S1-decorated actin filaments are highlighted in yellow. Jasplakinolide treatment did not significantly change actin-filament distribution. The scale bar represents 0.5 μm. (E) Quantification of the actin-filament density by measuring the length of actin filaments in electron micrographs of control nontreated and jasplakinolide-treated cells. Error bars represent SEM for measurements in ten different cells. Jasplakinolide treatment does not significantly affect the density of actin filaments. See also Movies S7 and S8. Current Biology 2008 18, 1581-1586DOI: (10.1016/j.cub.2008.08.070) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Jasplakinolide Does Not Affect Myosin-Driven Movement of Isolated Pigment Granules along Actin Filaments Examined in In Vitro Motility Assay (A) Successive images of fluorescently labeled actin filaments and pigment granules acquired in the absence (top panel) or in the presence (bottom panel) of jasplakinolide (1 μM). In both control and jasplakinolide-containing samples, pigment granules often attached to actin filaments and moved along them, thereby indicating that jasplakinolide does not significantly inhibit myosin-based transport of pigment granules. Numbers indicate time in s. The scale bar represents 0.5 μm. (B) Frequency histograms of movement velocities of pigment granules along the actin filaments in vitro in the absence (left) or presence (right) of jasplakinolide. In both the presence or the absence of jasplakinolide, the movement velocities peaked at ∼3 μm/min, thereby indicating that the drug has no detectable effect on the activity of myosin Va. See also Movies S9 and S10. Current Biology 2008 18, 1581-1586DOI: (10.1016/j.cub.2008.08.070) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Role of Actin Dynamics in the Myosin-Based Transport of Pigment Granules Myosin Va moves pigment granule toward the plus ends of actin filaments, which grow at the same time. Therefore, growth of actin filaments should increase distance traveled by pigment granule along them. Current Biology 2008 18, 1581-1586DOI: (10.1016/j.cub.2008.08.070) Copyright © 2008 Elsevier Ltd Terms and Conditions