1. Volume 20, Issue 16, Pages 1423-1431 (August 2010)
Differential Regulation of Unconventional Fission Yeast Myosins via the Actin Track 
Joseph E. Clayton, Matthew R. Sammons, Benjamin C. Stark, Alex R. Hodges, Matthew Lord 
Current Biology 
Volume 20, Issue 16, Pages (August 2010)
DOI: /j.cub
Copyright © 2010 Elsevier Ltd Terms and Conditions

2. Current Biology 2010 20, 1423-1431DOI: (10.1016/j.cub.2010.07.026)
Copyright © 2010 Elsevier Ltd Terms and Conditions

3. Figure 1
Differential Regulation of Fission Yeast Unconventional Myosins by Tropomyosin
(A–C) Actin-activated Mg2+-ATPase activity of Myo1p (A), Myo51p (B), and Myo52p (C) was measured as a function of actin (○) or actin-Cdc8p (●) concentration. Basal Myo2p ATPase activities (detected from control reactions lacking actin or actin-Cdc8p) and background Pi from actin or actin-Cdc8p (determined from controls lacking myosin) were subtracted from all measurements. Plots were generated from average values (±standard deviation [SD]) obtained from four different data sets. Curves were fit to Michaelis-Menten kinetics using KaleidaGraph software. Actin filaments were preincubated with Cdc8p at a concentration of 2:1 actin:Cdc8p molecules to ensure saturation of filaments with Cdc8p as previously described [24].
(D) Histograms summarizing average rates (±SD) of Myo1p, Myo51p, Myo52p, and myosin-Va motility for actin alone (□) and Cdc8p-bound filaments (■) (n = 60 filaments per experiment).
(E) Myo52p duty ratios in the presence of actin versus Cdc8p-bound actin. Data from plots of motility rate against number of motors available were fit to the equation V = (a × VMAX) × (1 − [1 − f]N), where V is actin filament velocity, VMAX is maximum filament velocity, f is duty ratio, N is total number of motors capable of interacting with the actin filament, and (a × VMAX) relates to the ability to move filaments at low motor density [50]. Fitting by nonlinear regression allows the duty ratio (f) to be determined as a parameter of the fit (see Supplemental Experimental Procedures for details regarding this experiment).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

4. Figure 2
Tropomyosin Blocks Interactions between Myo1p and Actin Filaments
(A) Coomassie blue-stained SDS-PAGE gel of a Myo1p-GFP sample that was one-step purified from myo1-GFP fission yeast cell extracts. Myo1p-GFP was isolated via GST-tagged forms of its light chains Cam1p and Cam2p.
(B) Control images of rhodamine-labeled actin filaments under TRITC versus GFP filters.
(C) Representative images from actin-Myo1p decoration experiments. Decoration of actin filaments alone (top) versus Cdc8p-bound actin filaments (bottom) by Myo1p-GFP is shown. Experiments were performed in the absence of nucleotide (strong actin-binding conditions). Insets (top) represent control experiments performed in the presence of 1 mM ATP (weak actin-binding conditions).
(D) Histograms quantifying the average GFP signals detected on actin or actin-Cdc8p filaments incubated with GFP-labeled Myo1p. GFP signals were scored relative to the corresponding rhodamine fluorescence intensity values. Measurements (±SD) were derived from experiments performed in the absence or presence of ATP (n = 40 filaments or filament clusters per experiment).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

5. Figure 3
Tropomyosin and Fimbrin Have Contrasting Effects on Myo1p Patch Lifetimes
(A) Differential interference contrast and epifluorescence images showing Myo1p-GFP localization in wild-type and cdc8-27 cells grown at 25°C. Fluorescence images are maximal projections generated from Z stacks (6 × 0.75 μm). Scale bar represents 4 μm.
(B) Myo1p patch intensities were tracked over time in wild-type (top) and cdc8-27 (center) cells. Images used were generated as described in (A). Average patch lifetimes (n = 30 patches) for the two strains are indicated on the plots (and are significantly different based on a paired Student's t test, p < ). The bottom plot shows the average patch intensities over time for the two data sets. Values were normalized by setting the highest average value to 1. The montages shown to the right of wild-type and cdc8-27 plots chart the lifetime of representative Myo1p patches (±SD, at 3 s intervals; scale bars represent 4 μm).
(C) Epifluorescence images showing Myo1p-GFP localization in wild-type, fim1Δ, and fim1Δ cdc8-27 cells grown at 25°C. Images are maximal projections generated from Z stacks. Scale bar represents 4 μm.
(D) Montage charting the lifetime of a representative Myo1p patch from a fim1Δ strain (3 s intervals; scale bar represents 4 μm).
(E) Myo1p patch intensities tracked over time in wild-type (top, n = 30) and fim1Δ (center, n = 45) cells. Average patch lifetimes (±SD) for the two strains are indicated on the plots (and are significantly different, p < ). The bottom plot shows average patch intensities over time for the two data sets (normalized as in B).
(F) Montage charting the lifetime of a representative Myo1p patch from a fim1Δ cdc8-27 strain (3 s intervals; scale bar represents 4 μm).
(G) Myo1p patch intensities tracked over time in wild-type (top, n = 30) and fim1Δ cdc8-27 (center, n = 50) cells. Average patch lifetimes for the two strains are indicated on the plots. Compared with cdc8-27 and fim1Δ single mutants, deviation from wild-type lifetimes (±SD) was much less significant in the fim1Δ cdc8-27 double mutant (p = 0.011). The bottom plot shows average patch intensities over time for the two data sets (normalized as in B). Note that time-lapse images of Myo1p patch dynamics captured for each of these three mutants (cdc8-27, fim1Δ, and cdc8-27 fim1Δ) were each performed in parallel with a wild-type control (recorded in an identical fashion) to account for any variability in the microscope settings or output. Fluorescence intensity values are arbitrary and should only be compared within parallel experiments.
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

6. Figure 4
Fimbrin Releases Myo1p Motors from Tropomyosin-Mediated Inhibition
(A) Representative images from actin-Myo1p decoration experiments. Decoration of actin-Fim1p filaments alone (top) versus actin filaments preincubated with both Fim1p and Cdc8p (bottom) is shown. Experiments were performed in the absence of nucleotide (strong actin-binding conditions). Insets represent control experiments performed in the presence of 1 mM ATP (weak actin-binding conditions).
(B) Histograms quantifying the average relative GFP signals detected on actin-Fim1p and actin-Cdc8p/Fim1p filaments incubated with GFP-labeled Myo1p. Average Myo1p-GFP signals (±SD) detected at actin and actin-Cdc8p filaments (taken from Figure 2D) are included for reference. GFP signals in these experiments were scored relative to the corresponding rhodamine fluorescence intensity values. Measurements were derived from experiments performed in the absence or presence of ATP (n = 40 filaments/filament clusters per experiment).
(C) Actin-activated ATPase assays comparing the motor activity (±SD) of Myo1p in the presence or absence of Fim1p- and Cdc8p-bound actin filaments. Assays were performed as described in Figure 1A using final protein concentrations of 10 μM actin, 5 μM Cdc8p, and 2.5 μM Fim1p.
(D) Panels show representative montages of rhodamine-labeled actin from in vitro motility movies with Myo1p. Top left: actin alone; bottom left: actin plus 2 μM Cdc8p and 0.4 μM Fim1p; top right: actin plus 2 μM Cdc8p and 0.4 μM Fim1-A2p. The plots on the bottom right compare motility rates (±SD) and the relative frequency of motile events observed for actin alone, actin + Fim1-A2p, and actin + Fim1-A2p and inhibitory Cdc8p.
(E) Actin-activated ATPase assays comparing the motor activity (±SD) of Myo2p in the presence of actin versus Fim1p-bound actin (protein concentrations: 10 μM actin, 2.5 μM Fim1p).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

7. Figure 5
Tropomyosin Promotes Directed Movement of Myo52p along Actin Cables
(A) Time-lapse montages showing directed movement of Myo52p-3xGFP particles toward the division site (top) or cell tip (bottom) within the same wild-type cell, captured using total internal reflection fluorescence microscopy. Panels to the far right are maximum projections summarizing particle trajectories.
(B) Plots comparing the average (±SD) speeds (n = 60), run lengths (n = 60), and frequencies (n = 270) of Myo52p-directed movement in wild-type versus cdc8-27 cells grown and monitored at 25°C. Frequency: number of directed Myo52p particle movements per cell (per 30 s movie). Paired Student's t tests were used to assess differences in average values (p < 0.05 indicates significant difference between data sets). Wild-type versus cdc8-27 cells: speed, p = 0.08; run length, p = 0.66; frequency, p =
(C) As in (B), except cells were grown and monitored at 30°C. Wild-type versus cdc8-27 cells: speed, p < ; run length, p = ; frequency, p < Run length data was also compared using the Kolmogorov-Smirnov test, which does not assume a Gaussian distribution (Figure S5).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

8. Figure 6
Schematic Model of Myosin Motor Regulation by Tropomyosin and Fimbrin at Fission Yeast Actin Structures
Endocytic patches: high concentrations of fimbrin prevent tropomyosin from binding to the branched actin network, limiting tropomyosin-mediated inhibition of myosin-I (Myo1p). Cables: tropomyosin favors myosin-V (Myo52p)-directed motility along the length of unbranched actin cables. Contractile rings: ring assembly relies on the compaction of myosin-II (Myo2p) nodes and unbranched actin filaments. Tropomyosin favors actomyosin interactions and ring assembly.
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions