FIGURE S4 â Histograms of stepping orientation of myosin Va and VIIa. 2. ..... (n=101) for stress fibers, lamellipodia and filopodia, respectively. The solid lines ...
Human Myosin VIIa Is an Extremely Slow Processive Motor Moving on Various Cellular Actin Structures
Osamu Satoshi Tsuyoshi Sakai*, Yoshikazu Tsukasaki*† , Ryosuke Tanaka‡, Takeomi Mizutani§, Tomonobu M. Watanabe||, Reiko Ikebe*, and Mitsuo Ikebe*
From *Department of Cellular and Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708; †Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612； ‡Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan; §Department of Advanced Transdisciplinary Sciences, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan; ||Laboratory for Comprehensive Bioimaging, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan.
Sato*,
Komatsu*,
1. Supplemental Figures & the Figure Legends FIGURE S1 – Calmodulin content of myosin VIIA FIGURE S2 – Calibration of TIRF microscope FIGURE S3 – Stepping orientation of myosin Va movements FIGURE S4 – Histograms of stepping orientation of myosin Va and VIIa 2. Legends to Supplemental Movies Supplemental Movie 1 – Myosin VIIa movement on single actin filaments. Supplemental Movie 2 – Myosin VIIa movement on demembranated MEF-3T3 cells. Supplemental Movie 3 – Myosin VIIa movement at the cell periphery on demembranated MEF-3T3 cells.
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FIGURE S1. Calmodulin content of myosin VIIa. To determine the numbers of CaM bound to HM7AΔTail and HM7AΔTail/LZ, we co-precipitated HM7AΔTail or HM7AΔTail/LZ with F-actin, and the pellets were subjected to SDS-PAGE as described in “Experimental Procedures.” Shown are typical SDS-PAGE images of HM7AΔTail and HM7AΔTail/LZ (stained with Coomassie Brilliant Blue G-250). The positions of M7HC (HM7AΔTail or HM7AΔTail/LZ heavy chain), actin, and CaM bands were shown in the left. The positions of molecular weight markers were shown between the gel images.
FIGURE S2
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&'()*+,-(./ FIGURE S2. Calibration of TIRF microscope. (A) Typical data for the stage calibration. (Left) 10 nm and (Right) 100 nm were shown. The position of FluoSpheres sulfate (0.2 µm) fluorescence was captured with TIRF microscope, and repeatedly moved the Proscan III stage at 10 and 100 nm withmicroscope 5 seconds interval. (B) Length vs. pixel relationship. Experiment was Fig.nm S1 Calibration of TIRF done as in A at 10 nm (n=22), 20 nm (n=18), 40 nm (n=23), 60 nm (n=23), 80 nm (n=24), and 100 nm (n=24) and pixel vs. length relationship was plotted. The error bars are s.d.
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FIGURE S3. Stepping orientation of myosin Va movements. (A) The typical stepping traces of myosin Va movement in (a) stress fibers, (b) lamellipodia, and (c) filopodia. (B) The polar plots of the individual stepping orientation of myosin Va HMM on (a) stress fibers, (b) lamellipodia and (c) filopodia. The stepping orientation of mouse myosin Va was measured and plotted as described in “Experimental Procedures.” The 0º is the orientation of individual myosin Va steppings, parallel to the moving direction.
g. S2 Trajectory ond Polarity of myosin Va stepping on stress fiber, lamellipodia and filopodia
FIGURE S4
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FIGURE S4. Histograms of stepping orientation of myosin Va and VIIa. (A–C) The histograms of stepping orientation of myosin Va HMM on (A) stress fibers, (B) lamellipodia and (C) filopodia. Data in Fig. S3 were re-plotted to calculate standard deviation. The histogram showed -3.4 ± 40.5º (mean ± s.d., n=132), -5.6 ± 36.7º (n=105) and 2.5 ± 36.1º (n=101) for stress fibers, lamellipodia and filopodia, respectively. The solid lines were best fit to Gaussian distributions. (D–F) The histogram of stepping orientation of HM7AΔTail/LZ on (D) stress fibers, (E) lamellipodia and (F) filopodia. Data in Fig. 5C were re-plotted, and the histogram showed -1.0 ± 61.8º (mean ± s.d., n=274), 0.5 ± 47.3º (n=263) and -1.9 ± 35.4º (n=270) for stress fibers, lamellipodia and filopodia, respectively.
LEGENDS TO SUPPLEMENTAL MOVIES
Supplemental Movie 1. Myosin VIIa movement on single actin filaments. The movement of HM7AΔTail/LZ on single actin filaments was observed using IX83-based TIRF microscope system as described in “Experimental Procedures.” Rhodamine-phalloidin labeled F-actin was used in this experiment. The movie was captured at the frame rate of 2 fps. The time was shortened X100 using Adobe Premiere software because of extremely slow velocity of myosin VIIa (~11 nm/s). The time line was shown in the movie (~8 min). The length of one side of the movie = 34 µm.
Supplemental Movie 2. Myosin VIIa movement on demembranated MEF-3T3 cells. MEF-3T3 cells were demembranated with Triton X-100, stained with Alexa Fluor 568 phalloidin, blocked with casein, and the movement of HM7AΔTail/LZ-Qdot 525 was observed using IX83-based TIRF microscope system as described in “Experimental Procedures.” The red signal of Alexa 568 and concomitant green signal of Qdot 525 were captured at the frame rate of 4 fps, and shortened the time X50. The movie shows the movements of HM7AΔTail/LZ-Qdot 525 on stress fibers, lamellipodia and filopodia. Note that myosin VIIa moves more slowly with a shorter run length in stress fibers. The velocities & run length of myosin VIIa on stress fibers, lamellipodia and
filopodia were 6.6 nm/s & 0.41 µm, 8.1 nm/s & 0.59 µm and 9.5 nm/s & 0.69 µm, respectively (see text). The length of one side of the movie = 2.6 µm.
Supplemental Movie 3. Myosin VIIa movement at the cell periphery on demembranated MEF-3T3 cells. The signals of Alexa 568 and Qdot 525 were simultaneously captured using single camera. The movie was captured at frame rate of 10 fps, and shortened the time X10. The length of one side of the movie = 27.6 µm.
