JCS100032 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. GFP-supervillin localizes preferentially to successor podosomes. Confocal immunofluorescence micrographs of a 7d cultured primary human macrophage expressing GFP-supervillin (A, green) and stained with anti-vinculin antibody (B, red), with merged images shown in (C). (D-F) An enlargement of the area denoted by the white box in (C). (G) Plots of fluorescence intensity for a single podosome. Note that maximal fluorescence intensities for vinculin do not coincide with those for GFP-supervillin. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Gelsolin shows no differential localization to podosome subpopulations. Confocal immunofluorescence micrographs of a 7d cultured primary human macrophage expressing GFP-supervillin (A, green), and stained for gelsolin using specific primary antibody (B, red) and for F-actin using Alexa Fluor 647-labeled phalloidin (C, blue). (D) Merged image of (A) and (B). (E) Merged image of (A), (B) and (C). Note that gelsolin is present at all F-actin-stained podosomes in the cell (magenta). Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Endogenous supervillin at successor and precursor podosomes. (A-I) Confocal immunofluorescence micrographs of 7d cultured primary human macrophages, stained for endogenous supervillin (A,D,G; green) and F-actin (B,E,H; red), with merged images shown in (C,F,I). Randomly chosen successor (white circles) and precursor podosomes (magenta circles) were analyzed for their respective supervillin content by measuring fluorescence intensities. Scale bars: 10 µm. (J) Analysis of supervillin-based fluorescence intensities in successor and precursor podosomes. For each value, 40 successor or 40 precursor podosomes were analyzed (4 cells from 4 different donors). For specific values, see supplementary material Table S1. *P<0.05, as determined by Mann-Whitney test.

• Supplemental Figure S4 -

  Fig. S4. Podosome numbers in macrophages transformed with various supervillin constructs. Macrophages were scored in groups according to podosome numbers. For each value and time, 30 randomly chosen cells from 3 independent experiments were evaluated. Values are given as mean percentage ± SEM of total cell counts (see also supplementary material Table S1; Fig. 4). *P<0.05; **P<0.01.

• Supplemental Figure S5 -

  Fig. S5. Overexpression of GFP-SV 1-174 induces the formation of myosin IIA-rich cables. Confocal immunofluorescence micrographs of a 7d cultured primary human macrophage expressing GFP-SV 1-174 (A) and stained for myosin IIA (B); merge shown in (C). Scale bar: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Subcellular localization of supervillin deletion mutants. Confocal immunofluorescence micrographs of 7d cultured primary human macrophages expressing supervillin constructs: a construct comprising the first F-actin binding region (SV 174-343; A-C), a construct comprising the second F-actin binding region (SV 343-570; D-F), a construct comprising the third F-actin binding region (SV 571-830; G-I), a C-terminal construct (SV 830-1072; J-L), or a C-terminal construct lacking the nuclear localization signal (SV 1010-1072; M-O). GFP signals in green (A,D,G,J,M), F-actin staining in red (B,E,H,K,N), merge in (C,F,I,L,O). Scale bars: 10 µm. Note the presence at both precursor and successor podosomes of all constructs containing an actin-binding region.

• Supplemental Figure S7 -

  Fig. S7. The SV 174-343 region competes with full-length supervillin for binding to podosomes. Confocal micrographs of macrophages expressing mRFP-supervillin (B,F), together with GFP-SV 174-343 (C) or GFP as control (G), and stained for F-actin (A,E); merges shown in (D,H). Note the retention of mRFP-supervillin on actin cables at the cell center (B) and its loss from podosomes (F) upon overexpression of GFP-SV 174-343. Scale bars: 10 µm.

• Supplemental Figure S8 -

  Fig. S8. RNAi-induced knockdown of supervillin, myosin IIA and L-MLCK. Western blots of lysates from primary human macrophages (A left,B,C,D,E) or U2OS cells (A right), treated with the indicated siRNA and developed with antibodies against (A,B) supervillin (H340 or HSV715 antibody, as indicated); (C) myosin IIA; (D) MLCK; or (E) phosphorylated myosin light chain (pMLC). Lines in A, right indicate that lanes were not directly adjacent on original blots. Molecular masses of markers in kDa are indicated.

• Supplemental Figure S9 -

  Fig. S9. Evaluation of podosome numbers following knockdown of supervillin, myosin IIA or gelsolin. Podosome numbers in 7d old macrophages, transfected with luciferase siRNA as control, or with supervillin-, myosin IIA- or gelsolin-specific siRNA or a combination thereof, as indicated. (A) Untransfected cells were scored in groups according to podosome numbers. For each value, 3x30 cells from 3 different donors were evaluated. For specific values, see supplementary material Table S1. (B,C) Macrophages expressing GFP-Lifeact were transfected with specific siRNAs, as indicated, and scored for (B) the average number of podosomes/cell and (C) the average change in the number of podosomes/cell at the indicated time (n=9 in all cases). For specific values, see supplementary material Table S1.

• Supplemental Figure S10 -

  Fig. S10. L-MLCK-GFP localizes to all podosomes. Confocal micrographs of polarized (A-D) and unpolarized (E-H) macrophages expressing GFP-L-MLCK (A,E) and mRFP-supervillin (B,F) counter-stained for F-actin with Alexa Fluor 647-coupled phalloidin (C,G); merged images (D,H). Note that GFP-L-MLCK is present at both precursor and successor podosomes, whereas mRFP-supervillin localizes preferentially to successor podosomes.

• Supplemental Table S1 -
• Movie 1 -

  Movie 1. GFP-supervillin localizes at a cap structure on podosome cores. 3D reconstruction showing a single podosome of a cell expressing GFP-supervillin (green), stained for F-actin (red) and vinculin (blue). Cell was imaged using a Leica SP-2 confocal microscope. Z stacks were taken from the bottom to the top of the cell with a step size of 0.1µm. Image processing was done using Volocity software (Improvision). Cell was cropped to the level of a single podosome and visualized in 3-D using the �3-D opacity� option. Note the cap-like decoration of GFP-supervillin at the F-actin rich podosome core and the typical ring-like decoration of vinculin. The 3-D image was tilted along various axes and rendered into a movie. Frame rate=15 frames/sec. For still images see Fig. 1K−M.

• Movie 2 -

  Movie 2. Dissolving podosomes acquire GFP-supervillin. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-supervillin (green, left channel), m-RFP actin (red, middle channel), and merge (right channel). Region of interest from cell, as indicated in Fig. 2A. Note also that disappearance of the mRFP-signal at podosomes is coupled with enrichment of GFP-supervillin. Exposure time: 500 ms, images were taken with a delay of 10 s between frames and are played back at 5 frames/s. Total elapsed time during experiment: 965.5 s, image width: 85 µm. For still images, see Fig. 2B−M.

• Movie 3 -

  Movie 3. Dissolving podosomes acquire GFP-supervillin. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-supervillin (green, left channel), Lifeact-RFP (red, middle channel), and merge (right channel). Note that disappearance of the RFP-signal at podosomes is coupled with enrichment of GFP-supervillin. Exposure time: 569 ms, images were taken with a delay of 30 s between frames and are played back at 2 frames/s. Total elapsed time during experiment: 600 s, image width: 35 µm. For still images, see Fig. 2N−Y.

• Movie 4 -

  Movie 4. GFP-myosin IIA contacts dissolving podosomes. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-myosin IIA (green) and mRFP-supervillin (red). Note formation of GFP-myosin IIA-enriched trailing edge and absence of mRFP-supervillin from podosomes at the leading edge (lower right). Contact of GFP-myosin IIA with mRFP-positive podosomes at the rear of the podosome field precedes their disappearance, while podosomes at the forward side of the field acquire mRFP-supervillin. Exposure time: 1 s for 488 nm channel, 3 s for 568 nm channel, with 2x binning; images were taken with a delay of 10 s between frames and are played back at 10 frames/s. Total elapsed time during experiment: 788.1 s, image width: 73.6 µm. For still images, see Fig. 6I−L.

• Movie 5 -

  Movie 5. Supervillin-containing podosomes are connected by contractile GFP-myosin IIA-positive cables. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-myosin IIA (green) and mRFP-supervillin (red). Podosomes at the rear of the podosome field (right) are connected and pulled upon by GFP-myosin-IIA-positive cables. Exposure time: 1 s for 488 nm channel, 3 s for 568 nm channel, with 2x binning; images were taken with a delay of 10 s between frames and are played back at 10 frames/s. Total elapsed time during experiment: 625.8 s, image width: 50.1 µm. For still image, see Fig. 6N.

• Movie 6 -

  Movie 6. Supervillin-containing podosomes are connected by contractile GFP-myosin IIA-positive cables. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-myosin IIA (green, left channel) and mRFP-supervillin (red, middle channel), and merge (right channel). Region of interest from supplementary material Movie 5, as indicated in Fig. 6N. Podosomes (from the rear of the podosome field in supplementary material Movie 4) are connected and pulled upon by GFP-myosin-IIA-positive cables. Exposure time: 1 s for 488 nm channel, 3 s for 568 nm channel, with 2x binning; images were taken with a delay of 10 s between frames and are played back at 10 frames/s. Total elapsed time during experiment: 625.8 s, image width: 85 µm. For still image, see Fig. 6O.

• Movie 7 -

  Movie 7. GFP-L-MLCK localises to successor and precursor podosomes. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-L-MLCK (green; left channel) and mRFP-supervillin (red; middle channel), and merge (right channel). GFP-L-MLCK localizes to both successor and precursor podosomes, whereas mRFP-supervillin is observed only at successor podosomes. Exposure time: 841 ms for 488 nm channel, 500 ms for 568 nm channel, with 1x binning; images were taken with a delay of 15 s between frames and are played back at 10 frames/s. Total elapsed time during experiment: 1837s, image width: 90 µm.

• Movie 8 -

  Movie 8. Cells expressing GFP SV 1-830 show formation of a compact myosin IIA ring around the podosome core. 3D reconstruction showing a single podosome in a macrophage expressing GFP-SV 1-830 (green) counter-stained for F-actin (red) and myosin IIA (white). Cell was imaged using a Leica SP-2 confocal microscope. Z stacks were taken from the bottom to the top of the cell with a step size of 0.1µm. Image processing was done using Volocity software (Improvision). Cells were cropped to the level of a single podosome and visualized in 3-D using the �3-D opacity� option. Note the tight ring-like decoration of myosin IIA around the podosome core. The 3-D image was tilted along various axes and rendered into a movie. Frame rate: 15 frames/sec. For still images see Fig. 7H−J.

• Movie 9 -

  Movie 9. Cells expressing GFP-supervillin show a more dispersed localization of myosin IIA around the podosome core. 3D reconstruction showing a single podosome in a macrophage expressing GFP-supervillin (green) counter-stained for F-actin (red) and myosin IIA (white). Cells were imaged using a Leica SP-2 confocal microscope. Z stacks were taken from the bottom to the top of the cell with a step size of 0.1 µm. Image processing was done using Volocity software (Improvision). Cells were cropped to the level of a single podosome and visualized in 3-D using the �3-D opacity� option. Note the dispersed ring-like decoration of myosin IIA around the podosome. The 3-D image was tilted along various axes and rendered into a movie. Frame rate: 15 frames/sec. For still images see Fig. 7K−M.

• Movie 10 -

  Movie 10. Precursor podosomes acquire GFP-supervillin on addition of blebbistatin. Confocal time-lapse video of a 7d cultured primary human macrophage expressing GFP-supervillin (green, left channel), Lifeact-RFP (red, middle channel), and merge (right channel). 2 µM blebbistatin was added 11 min after the start of the experiment Exposure time: 273 ms, images were taken with a delay of 60 s between frames and are played back at 3 frames/s. Total elapsed time during experiment: 94.24 minutes, image width: 65 µm.