SUPPLEMENTARY INFORMATION

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a 14 12 Densitometry (AU) 1 8 6 4 2 t b 16 NMHC-IIA GAPDH NMHC-IIB Densitometry (AU) 14 12 1 8 6 4 2 1 nm 1 nm 1 nm 1 nm sirna 1 nm 1 nm Figure S1 S4 Quantification of protein levels.

1 a Densitometry (AU) t b 16 NMHC-IIA GAPDH NMHC-IIB Densitometry (AU) nm 1 nm 1 nm 1 nm sirna 1 nm 1 nm Figure S1 S4 Quantification of protein levels. (a) The microtubule expansion in wild-type ES cells after bistatin treatment does not involve changes in the protein levels of tubulin. Wild-type ES cells treated with 25 nm bistatin () for 3 minutes showed immediate expansion of microtubules, but no increase in the total levels of -tubulin. (b) RNA interference by myosin IIA sirna transfection in human foreskin fibroblasts reduced protein levels of myosin IIA by 6% at 1 nm and 65% at 1 nm sirna (densitometry calculations were corrected for GAPDH levels). Myosin IIB levels were not significantly altered (right-hand graph). Error bars indicate range Nature Publishing Group

a 6 5 Velocity ( m/h) 4 3 2 1 WT IIB -/- fibroblasts WT IIB -/- ES cells b 12 1 IIA -/- ES cells c IIA -/- IIA -/- Velocity ( m/h) 8 6 4 2 5 1 Figure S2 S1 Analyses of roles of

(b, c) Inhibition of myosin IIB by bistatin () in myosin IIA-null ES cells.

2 a 6 5 Velocity ( m/h) WT IIB -/- fibroblasts WT IIB -/- ES cells b 12 1 IIA -/- ES cells c IIA -/- IIA -/- Velocity ( m/h) Figure S2 S1 Analyses of roles of myosin IIB. (a) Myosin IIB-deficient fibroblasts and ES cells exhibit slightly slower migration rates compared to wild-type cells, as quantified by time-lapse microscopy. (b, c) Inhibition of myosin IIB by bistatin () in myosin IIA-null ES cells. Blebbistatin (5 or 1 nm) treatment of myosin IIA-null ES cells (IIA-/-) to completely inhibit all myosin II isoforms (including IIB) did not significantly change migration rates (b), but 5 nm bistatin eliminated residual stress fibres (c) Nature Publishing Group

3 a 1 b D/T.6.4 D/T P =.31 WT IIA-/- P =.39 Figure S3 S2 Myosin II-deficient cells show decreased directionality. Human foreskin fibroblasts treated with bistatin (a) or myosin IIA-null ES cells (b) migrate with less directional persistence compared to their untreated or wild-type counterparts. Directionality was evaluated by time-lapse microscopy and calculated as D/T: the ratio between the total trajectory length (T) and the linear distance between the start and end points (D) Nature Publishing Group

4 Fluorescence intensity (AU) Fluorescence intensity (AU) Rac Distance (μm) Tiam Distance (μm) Figure S4 S3 Line scans of fluorescence intensity of the immunolocalization of Rac1 and Tiam1 at the leading edge. Fluorescence was measured by MetaMorph image analysis software starting at the cell periphery (left) along 7 lines drawn perpendicular to the leading edge of the myosin IIAnull ES cell in Fig. 4a. Fill colors designate paired scans for each protein. Note the strong, co-localized staining for Rac1 and Tiam1 near the leading edge Nature Publishing Group

5 Legends for Supplementary Videos The supplementary videos are best viewed with QuickTime 7 or a higher version. The viewer is available at no cost from Apple at (for Mac) or (for Windows). Supplementary videos 1-4: COS-7 cells, which lack the myosin IIA isoform but express myosin IIB, were co-transfected with mcherry- -tubulin and either EGFPmyosin IIA or EGFP-myosin IIB plasmids. Time-lapse spinning-disk focal fluorescent imaging was performed hours after transfection for ~6 minutes. Human foreskin fibroblasts, which express both myosin II isoforms, were transfected and imaged similarly. Video movies were compressed using QuickTime Pro and the H.264 codec; the original higher-resolution movies can be obtained from the authors upon request. Supplementary video 1: Myosin IIA-transfected COS-7 cells show substantial microtubule dynamics (left panel and red in the merged color image) in areas of active lamellar ruffling. EGFP-myosin IIA (middle, green) shows vigorous retrograde movements of myosin IIA clusters in lamellae. The scale bar represents 5 μm. Supplementary video 2: Myosin IIB-transfected COS-7 cells show relatively stable microtubules (left, red) with slow turnover. The EGFP-myosin IIB (middle, green) was primarily associated with stress fibers, and the low quantities in lamellae displayed slower retrograde flow than myosin IIA. Similar stability of microtubules was observed in cells without myosin II transfection. The scale bar represents 5 μm. Supplementary video 3: Human fibroblasts transfected with mcherry- -tubulin (left, red) and EGFP-myosin IIA (middle, green) show microtubule dynamics and vigorous retrograde flow of the EGFP-myosin IIA. The scale bar represents 5 μm. Supplementary video 4: Human fibroblasts were transfected with mcherry- tubulin (left, red) and EGFP-myosin IIB (middle, green) and show microtubule dynamics (these cells express endogenous myosin IIA) but only minimal retrograde flow of the EGFP-myosin IIB. The scale bar represents 5 μm. Supplementary video 5: Comparisons of microtubule dynamics in COS-7 cells transfected with myosin IIA (left panels), trol (center), or myosin IIB (right) plasmids. The scale bar represents 5 μm Nature Publishing Group

6 Supplemental Information: Equipment and Settings Time-Lapse Fluorescence Microscopy: Dual wavelength time-lapse images of mcherry-tubulin with EGFP-myosin IIA or EGFP-myosin IIB were acquired using an Ultraview ERS spinning-disk focal scanning system (Perkin-Elmer) attached to a Zeiss Axiovert 2M microscope equipped with a 1X Plan-APOCHROMAT (N.A. 1.4) oil DIC objective (Zeiss). 24-hours after transfection cells were plated onto glass-bottom culture dishes (MatTek). Images were captured every 6 seds (4-5 sec. dead time) for total durations of approximately 6 minutes using a Hamamatsu EM CCD camera (Hamamatsu Corporation) in 1x1 binning mode with a center sub-pixel array of 496x496. A Melles Griot Kr-Ar laser was used for excitation of EGFP and mcherry chimeric proteins at 488 nm and 568 nm, respectively. Exposure times and laser power were ~5ms and 35% for the 488 nm line and ~75 ms and 55% for the 568 nm line. An environmental chamber mounted on the microscope maintained stant 37ºC temperature, ~1% CO2, and humidity during acquisition. Image filtering and quantification: No image filtering was performed on fixed samples. Sequential background subtraction, low pass filtering, Gaussian filtering and sharpening image processing was carried out for fluorescence time series using custom-made volution filters created on both ImageJ (NIH) and MetaMorph software (Molecular Devices). All volution filter settings were the same for all images collected. Images were acquired at a bit-depth of 16 and then verted to 8-bit format following filtering. For analysis of myosin IIA and IIB flow rates within the lamella, kymographs of raw data were generated with ImageJ, and the slopes of lines depicting myosin flow were calculated from X, Y coordinates and verted to micrometers/minute. Microtubule dwell time was calculated from the time-lapse movie sequences by counting the number of frames in which the tip of a microtubule stayed within a zone 1 micrometers from the leading edge of cells Nature Publishing Group

JCB. Supplemental material THE JOURNAL OF CELL BIOLOGY. Prospéri et al.,

JCB. Supplemental material THE JOURNAL OF CELL BIOLOGY. Prospéri et al., Supplemental material JCB Prospéri et al., http://www.jcb.org/cgi/content/full/jcb.201501018/dc1 THE JOURNAL OF CELL BIOLOGY Figure S1. Myo1b Tail interacts with YFP-EphB2 coated beads and genistein inhibits