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Macrophage podosomes assemble at the leading lamella by growth and fragmentation.

Evans JG, Correia I, Krasavina O, Watson N, Matsudaira P - J. Cell Biol. (2003)

Bottom Line: The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum.Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures.Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: BioImaging Center, Cambridge, MA 02142, USA. jgevans@wi.mit.edu

ABSTRACT
Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

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Quantitative analysis of podosome assembly. (A) 2-D images of β-actin–EYFP fluorescence recorded by deconvolution confocal microscopy at 15-s intervals for 30 min. The top inset compares β-actin–EYFP fluorescence at t = 0 (green) and t = 30 (red) Bar, 3 μm. An x, y, t (red, purple, blue) boxed area (17.9 μm × 9.8 μm × 28.25 min) was analyzed by kymography. Side projections (bottom inset) of the kymograph show that the leading edge extends in the y-direction (purple axis) over time (blue axis). The boxed region in the bottom inset shows the position of the subregion (8.45 μm × 4.85 μm × 8.25 min) shown in the main panel. The kymograph displays the time-dependent location of podosomes as a centroid (yellow line) and volume (transparent green). Short-lived podosomes (arrows) and branched podosomes (arrowheads) are present. A 2-D image of the last time point forms the floor of the kymograph box (Video 3). (B) Branched podosomes (green) are primarily present at the extreme leading edge, whereas simple podosomes (red) are present throughout the leading edge adhesion zone (∼5 μm). The x, y, t volume (red, purple, blue) measures 17.9 μm × 9.8 μm × 5 min with the leading edge facing right. (C) In untreated macrophages, β-actin–EYFP FRAP occurred with a half time of 21.37 ± 3.26 s (filled circles, n = 7). No FRAP occurred in cells treated with jasplakinolide (filled squares, n = 8). Latrunculin A treatment did not prevent FRAP until podosomes were completely disassembled (filled diamonds, n = 8). Paclitaxel- and demecolcine-treated cells recovered fluorescence at similar rates to controls 21.24 ± 3.22 s (open squares, n = 8) and 16.38 ± 2.57 s (open diamonds, n = 8), respectively. Representative plots for each treatment are shown. (D) Selected frames of deconvolved β-actin–EYFP fluorescence from an untreated IC-21 macrophage immediately before (inset) and after photobleaching (green), and ∼2 min later (red). Green and red images are overlaid so that FRAP within the photobleached region (boxed) is indicated in red. Bar, 5 μm. (E) EM analysis of a detergent-extracted IC-21 macrophage. Podosomes (arrowhead) appeared in close proximity to microtubules (arrow) throughout the leading lamella. At higher magnification, microtubules (arrow) appeared to pass over or next to the tightly bundled podosome core (arrowhead). Bars: 5 μm (top inset), 500 nm (main panel), and 200 nm (bottom inset). (F and G) Kymographs from IC-21 macrophages treated with either 1 μM demecolcine for 5 min (F) or 10 μM paclitaxel for 45 min (G). Simple (arrows) and branched podosomes (arrowheads) are present in F and G, but traces appear shorter after demecolcine treatment and longer after paclitaxel treatment, compared with untreated cells (A). All dimensions as indicated for A. (H) Quantitation of centroid trace lengths expressed as the mean lifetime of podosomes from untreated (n = 5), demecolcine-treated (n = 5), and paclitaxel-treated (n = 5) cells for simple and branched podosomes (left). Branched podosome lifetime is shown per daughter (i.e., branch) and for the total length (from appearance of first daughter until disassembly of last daughter). The fusion and fission events (right) were identified and expressed as a rate (events/min).
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fig4: Quantitative analysis of podosome assembly. (A) 2-D images of β-actin–EYFP fluorescence recorded by deconvolution confocal microscopy at 15-s intervals for 30 min. The top inset compares β-actin–EYFP fluorescence at t = 0 (green) and t = 30 (red) Bar, 3 μm. An x, y, t (red, purple, blue) boxed area (17.9 μm × 9.8 μm × 28.25 min) was analyzed by kymography. Side projections (bottom inset) of the kymograph show that the leading edge extends in the y-direction (purple axis) over time (blue axis). The boxed region in the bottom inset shows the position of the subregion (8.45 μm × 4.85 μm × 8.25 min) shown in the main panel. The kymograph displays the time-dependent location of podosomes as a centroid (yellow line) and volume (transparent green). Short-lived podosomes (arrows) and branched podosomes (arrowheads) are present. A 2-D image of the last time point forms the floor of the kymograph box (Video 3). (B) Branched podosomes (green) are primarily present at the extreme leading edge, whereas simple podosomes (red) are present throughout the leading edge adhesion zone (∼5 μm). The x, y, t volume (red, purple, blue) measures 17.9 μm × 9.8 μm × 5 min with the leading edge facing right. (C) In untreated macrophages, β-actin–EYFP FRAP occurred with a half time of 21.37 ± 3.26 s (filled circles, n = 7). No FRAP occurred in cells treated with jasplakinolide (filled squares, n = 8). Latrunculin A treatment did not prevent FRAP until podosomes were completely disassembled (filled diamonds, n = 8). Paclitaxel- and demecolcine-treated cells recovered fluorescence at similar rates to controls 21.24 ± 3.22 s (open squares, n = 8) and 16.38 ± 2.57 s (open diamonds, n = 8), respectively. Representative plots for each treatment are shown. (D) Selected frames of deconvolved β-actin–EYFP fluorescence from an untreated IC-21 macrophage immediately before (inset) and after photobleaching (green), and ∼2 min later (red). Green and red images are overlaid so that FRAP within the photobleached region (boxed) is indicated in red. Bar, 5 μm. (E) EM analysis of a detergent-extracted IC-21 macrophage. Podosomes (arrowhead) appeared in close proximity to microtubules (arrow) throughout the leading lamella. At higher magnification, microtubules (arrow) appeared to pass over or next to the tightly bundled podosome core (arrowhead). Bars: 5 μm (top inset), 500 nm (main panel), and 200 nm (bottom inset). (F and G) Kymographs from IC-21 macrophages treated with either 1 μM demecolcine for 5 min (F) or 10 μM paclitaxel for 45 min (G). Simple (arrows) and branched podosomes (arrowheads) are present in F and G, but traces appear shorter after demecolcine treatment and longer after paclitaxel treatment, compared with untreated cells (A). All dimensions as indicated for A. (H) Quantitation of centroid trace lengths expressed as the mean lifetime of podosomes from untreated (n = 5), demecolcine-treated (n = 5), and paclitaxel-treated (n = 5) cells for simple and branched podosomes (left). Branched podosome lifetime is shown per daughter (i.e., branch) and for the total length (from appearance of first daughter until disassembly of last daughter). The fusion and fission events (right) were identified and expressed as a rate (events/min).

Mentions: Branches in the kymographs showed qualitatively that podosomes may arise by fission of older podosomes. Using actin as a marker of podosomes, we quantified the assembly and turnover dynamics of the entire population of podosomes in the leading lamella by measuring their xy position and lifetime using kymographs (Fig. 4 A and Video 4). Analysis of kymographs revealed two podosome populations; short-lived “simple” and longer-lived “branched” podosomes. Simple podosomes correspond to the short tracks that assembled de novo and disappeared within a minute. In contrast, tracks of the majority of podosomes were long and continuous with average lifetimes of 7 min (Fig. 4, A and H).


Macrophage podosomes assemble at the leading lamella by growth and fragmentation.

Evans JG, Correia I, Krasavina O, Watson N, Matsudaira P - J. Cell Biol. (2003)

Quantitative analysis of podosome assembly. (A) 2-D images of β-actin–EYFP fluorescence recorded by deconvolution confocal microscopy at 15-s intervals for 30 min. The top inset compares β-actin–EYFP fluorescence at t = 0 (green) and t = 30 (red) Bar, 3 μm. An x, y, t (red, purple, blue) boxed area (17.9 μm × 9.8 μm × 28.25 min) was analyzed by kymography. Side projections (bottom inset) of the kymograph show that the leading edge extends in the y-direction (purple axis) over time (blue axis). The boxed region in the bottom inset shows the position of the subregion (8.45 μm × 4.85 μm × 8.25 min) shown in the main panel. The kymograph displays the time-dependent location of podosomes as a centroid (yellow line) and volume (transparent green). Short-lived podosomes (arrows) and branched podosomes (arrowheads) are present. A 2-D image of the last time point forms the floor of the kymograph box (Video 3). (B) Branched podosomes (green) are primarily present at the extreme leading edge, whereas simple podosomes (red) are present throughout the leading edge adhesion zone (∼5 μm). The x, y, t volume (red, purple, blue) measures 17.9 μm × 9.8 μm × 5 min with the leading edge facing right. (C) In untreated macrophages, β-actin–EYFP FRAP occurred with a half time of 21.37 ± 3.26 s (filled circles, n = 7). No FRAP occurred in cells treated with jasplakinolide (filled squares, n = 8). Latrunculin A treatment did not prevent FRAP until podosomes were completely disassembled (filled diamonds, n = 8). Paclitaxel- and demecolcine-treated cells recovered fluorescence at similar rates to controls 21.24 ± 3.22 s (open squares, n = 8) and 16.38 ± 2.57 s (open diamonds, n = 8), respectively. Representative plots for each treatment are shown. (D) Selected frames of deconvolved β-actin–EYFP fluorescence from an untreated IC-21 macrophage immediately before (inset) and after photobleaching (green), and ∼2 min later (red). Green and red images are overlaid so that FRAP within the photobleached region (boxed) is indicated in red. Bar, 5 μm. (E) EM analysis of a detergent-extracted IC-21 macrophage. Podosomes (arrowhead) appeared in close proximity to microtubules (arrow) throughout the leading lamella. At higher magnification, microtubules (arrow) appeared to pass over or next to the tightly bundled podosome core (arrowhead). Bars: 5 μm (top inset), 500 nm (main panel), and 200 nm (bottom inset). (F and G) Kymographs from IC-21 macrophages treated with either 1 μM demecolcine for 5 min (F) or 10 μM paclitaxel for 45 min (G). Simple (arrows) and branched podosomes (arrowheads) are present in F and G, but traces appear shorter after demecolcine treatment and longer after paclitaxel treatment, compared with untreated cells (A). All dimensions as indicated for A. (H) Quantitation of centroid trace lengths expressed as the mean lifetime of podosomes from untreated (n = 5), demecolcine-treated (n = 5), and paclitaxel-treated (n = 5) cells for simple and branched podosomes (left). Branched podosome lifetime is shown per daughter (i.e., branch) and for the total length (from appearance of first daughter until disassembly of last daughter). The fusion and fission events (right) were identified and expressed as a rate (events/min).
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fig4: Quantitative analysis of podosome assembly. (A) 2-D images of β-actin–EYFP fluorescence recorded by deconvolution confocal microscopy at 15-s intervals for 30 min. The top inset compares β-actin–EYFP fluorescence at t = 0 (green) and t = 30 (red) Bar, 3 μm. An x, y, t (red, purple, blue) boxed area (17.9 μm × 9.8 μm × 28.25 min) was analyzed by kymography. Side projections (bottom inset) of the kymograph show that the leading edge extends in the y-direction (purple axis) over time (blue axis). The boxed region in the bottom inset shows the position of the subregion (8.45 μm × 4.85 μm × 8.25 min) shown in the main panel. The kymograph displays the time-dependent location of podosomes as a centroid (yellow line) and volume (transparent green). Short-lived podosomes (arrows) and branched podosomes (arrowheads) are present. A 2-D image of the last time point forms the floor of the kymograph box (Video 3). (B) Branched podosomes (green) are primarily present at the extreme leading edge, whereas simple podosomes (red) are present throughout the leading edge adhesion zone (∼5 μm). The x, y, t volume (red, purple, blue) measures 17.9 μm × 9.8 μm × 5 min with the leading edge facing right. (C) In untreated macrophages, β-actin–EYFP FRAP occurred with a half time of 21.37 ± 3.26 s (filled circles, n = 7). No FRAP occurred in cells treated with jasplakinolide (filled squares, n = 8). Latrunculin A treatment did not prevent FRAP until podosomes were completely disassembled (filled diamonds, n = 8). Paclitaxel- and demecolcine-treated cells recovered fluorescence at similar rates to controls 21.24 ± 3.22 s (open squares, n = 8) and 16.38 ± 2.57 s (open diamonds, n = 8), respectively. Representative plots for each treatment are shown. (D) Selected frames of deconvolved β-actin–EYFP fluorescence from an untreated IC-21 macrophage immediately before (inset) and after photobleaching (green), and ∼2 min later (red). Green and red images are overlaid so that FRAP within the photobleached region (boxed) is indicated in red. Bar, 5 μm. (E) EM analysis of a detergent-extracted IC-21 macrophage. Podosomes (arrowhead) appeared in close proximity to microtubules (arrow) throughout the leading lamella. At higher magnification, microtubules (arrow) appeared to pass over or next to the tightly bundled podosome core (arrowhead). Bars: 5 μm (top inset), 500 nm (main panel), and 200 nm (bottom inset). (F and G) Kymographs from IC-21 macrophages treated with either 1 μM demecolcine for 5 min (F) or 10 μM paclitaxel for 45 min (G). Simple (arrows) and branched podosomes (arrowheads) are present in F and G, but traces appear shorter after demecolcine treatment and longer after paclitaxel treatment, compared with untreated cells (A). All dimensions as indicated for A. (H) Quantitation of centroid trace lengths expressed as the mean lifetime of podosomes from untreated (n = 5), demecolcine-treated (n = 5), and paclitaxel-treated (n = 5) cells for simple and branched podosomes (left). Branched podosome lifetime is shown per daughter (i.e., branch) and for the total length (from appearance of first daughter until disassembly of last daughter). The fusion and fission events (right) were identified and expressed as a rate (events/min).
Mentions: Branches in the kymographs showed qualitatively that podosomes may arise by fission of older podosomes. Using actin as a marker of podosomes, we quantified the assembly and turnover dynamics of the entire population of podosomes in the leading lamella by measuring their xy position and lifetime using kymographs (Fig. 4 A and Video 4). Analysis of kymographs revealed two podosome populations; short-lived “simple” and longer-lived “branched” podosomes. Simple podosomes correspond to the short tracks that assembled de novo and disappeared within a minute. In contrast, tracks of the majority of podosomes were long and continuous with average lifetimes of 7 min (Fig. 4, A and H).

Bottom Line: The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum.Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures.Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: BioImaging Center, Cambridge, MA 02142, USA. jgevans@wi.mit.edu

ABSTRACT
Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

Show MeSH
Related in: MedlinePlus