supplementary information - media.nature.com · supplementary information 2 Figure S2 Rapid arrest of keratocytes by Triton-aldehyde mixture used to prepare cytoskeletons and cryo

s u p p l e m e n ta ry i n f o r m at i o n

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DOI: 10.1038/ncb2044

Figure S1 Overview images of negatively stained Triton/glutaraldehyde cytoskeletons used for tomography. a, goldfish (CAR) fibroblast; b, trout

keratocyte; c, Swiss 3T3 mouse fibroblast spreading on poly-L-lysine; d, B16F1 mouse melanoma cell. Bars: 10µm.

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Figure S2 Rapid arrest of keratocytes by Triton-aldehyde mixture used to prepare cytoskeletons and cryo-tomography of vitreously frozen cytoskeletons. a-c. Cells were plated onto formvar-coated coverslips and viewed by phase contrast microscopy; the growth medium was exchanged for the fixative mixture after the last live frame (b, time in secs) and the

fixed image (c) taken 5 secs later. d, the same group of cells in the EM after negative staining. e, Tomogram section of a keratocyte fixed as in c, but then rapidly frozen and imaged in ice. Image corresponds to an 8nm tomogram section. A scan through the tomogram is shown in Supplementary Information Movie 3 Bars: a-d, 30µm; e, 200nm.




Figure S3 Actin filament pairs in keratocyte lamellipodia. a, Tomogram section (0.75nm thick) of peripheral region (darkened area indicated in inset) of a negatively stained lamellipodium of a keratocyte fixed in an aldehyde/Triton mixture. b-d, sections (3.6 nm thick) at different

Z-levels from a tomogram of the lamellipodium tip of a keratocyte fixed in formaldehyde/glutaraldehyde, frozen and imaged in ice. White arrows indicate filament pairs. The tomograms were generated from tilt series around 2 axes in a and 1 axis in b-d. Bars, 200nm.




Figure S4 Actin filaments are continuous and unbranched in lamellipodia of Swiss 3T3 cells and B16F1 mouse melanoma cells. a, tomogram section (0.75nm) of lamellipodium front in a 3T3 fibroblast (for low magnification view, see Supplementary Information, Fig. S1c). b, c, two views of filament traces through the tomogram in a. A scan through the tomogram is shown in the Supplementary Information, Movie 6. d, plastic cross section of 3T3

cell lamellipodium. e, tomogram section (0.75nm) of lamellipodium front in a B16 melanoma cell (for low magnification view see Supplementary Information, Fig. S1d). f, g two views of filament traces through the tomogram in e. A scan through the tomogram is shown in the Supplementary Information, Movie 7. h, plastic cross section of B16 cell lamellipodium. Bars: a-c; e-g, 50nm; d, h, 250nm.




Figure S5 Actin filament densities in lamellipodia. a, illustration of procedure used for counting filaments. Image shows a section of the tomogram from Fig. 2f onto which a vertical plane 0.3µm long and traversing the depth of the tomogram was superimposed (yellow line) in this case at a distance of 200nm from the lamellipodium tip. At sequential

positions in Z, filaments that crossed the plane were marked by red lines and summed through the tomogram. Bar, 100nm. b, Collected data on filament densities for the four cell types studied. The numbers correspond to the average of 5 tomograms of negatively stained cytoskeletons from each cell type.




Figure S6 The Arp2/3 complex is retained in Triton/glutaraldehyde cytoskeletons. Images of a B16 melanoma cell transfected with p16ArpC5-GFP, before

(live) and after fixation (fixed) in the aldehyde/Triton mixture used to prepare cytoskeletons for electron tomography. Bar, 10µm.




Supplementary Movie Legends

Movie S1 Scan through tomogram and the filament model corresponding to the fish fibroblast lamellipodium in Fig. 1a-c. The scan starts at the ventral surface of the lamellipodium, moves in Z to the dorsal surface (marked by gold particles) and then returns to the ventral surface, with the tracked filaments superimposed, finally merging into the model. The tomogram was generated from a single axis tilt series.

Movie S2 Scan through tomogram and the filament model corresponding to fish fibroblast lamellipodium and microspike bundle in Fig. 2f, g. The scan starts on the support film close to the ventral surface, traverses to the dorsal surface in Z and returns to the ventral surface with the tracked filaments superimposed. In the last part of the video, most of the filaments have been deleted leaving some selected filaments (in purple) and the filament plus ends (yellow) to show the mixed orientation of filaments and the distribution of plus ends at the surface of the network. The tomogram was generated from a single axis tilt series.

Movie S3 Scan through tomogram of keratocyte cytoskeleton embedded in vitreous ice and corresponding to Fig. S2e. At the end of the scan in Z, filament tracks are superimposed and the model rotated to reveal filament arrangements.

Movie S4a Scan through tomogram corresponding to the keratocyte lamellipodium in Fig. 3a. The scan starts at the ventral surface and finishes at the dorsal surface. The blue rings correspond to hollow spheres (appearing as rings in the tomogram sections) placed at intersections where filaments overlap. The red rings correspond to hollow spheres marking sites where one filament appears to terminate at the side of another filament.

Movie S4b Scan of tomogram as in movie 4a but with the filament tracks (appearing as green dashes) superimposed to indicate tracking routine. The filament model (Fig. 3b) was created by joining the tracking points.

Movie S5 Scan through section of tomogram corresponding to Fig. 3c-g, showing overlapping events.

Movie S6 Scan through tomogram and the filament model of the lamellipodium of a 3T3 fibroblast corresponding to Supplementary Information, Fig. S4a. The scan starts at the dorsal surface of the lamellipodium network, moves in Z to the ventral surface. After zooming in, the tomogram is scanned once more and the tracked filaments superimposed, finally merging into the model. The tomogram was generated from tilt series around two axes.

Movie S7 Scan through tomogram and the filament model of the lamellipodium of a B16 melanoma cell corresponding to Supplementary Information, Fig. S4e. The scan starts at the dorsal surface of the lamellipodium network, moves in Z to the ventral surface. After zooming in, the tomogram is scanned once more and the tracked filaments superimposed, finally merging into the model. The tomogram was generated from tilt series around two axes.


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supplementary information - media.nature.com · supplementary information 2 Figure S2 Rapid arrest of keratocytes by Triton-aldehyde mixture used to prepare cytoskeletons and cryo