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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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Podosomes colocalize with integrins and are in close proximity to the substratum. (A) Maximum intensity projection of phalloidin-FITC fluorescence showing distribution of F-actin in an IC-21 macrophage. (B) Overlay of phalloidin-FITC (green) and anti-cd11c fluorescence (red and top inset). 3-D colocalization (yellow and bottom inset) of the two channels predominantly at the leading edge and actin foci. (C) Proximity of podosomes to the substratum as shown by IRM (top inset). Colocalization (yellow and bottom inset) of phalloidin-FITC with the inverted IRM signal shows leading edge and podosome-containing regions in close proximity to the substratum. (D) Podosomes show a high correlation of integrin colocalization (blue, bottom inset in B) and substratum proximity (red, bottom inset in C) as indicated by the white signal of the majority of podosomes. Bars, 10 μm. (E and F) 3-D reconstruction of the boxed region in D shows that F-actin (green) is present in large (arrow) and small (arrowhead) podosomes, and that both are in close proximity to the substratum as shown by IRM (red) and contain integrin colocalization (yellow). Grid squares are 1 μm × 1 μm.
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fig1: Podosomes colocalize with integrins and are in close proximity to the substratum. (A) Maximum intensity projection of phalloidin-FITC fluorescence showing distribution of F-actin in an IC-21 macrophage. (B) Overlay of phalloidin-FITC (green) and anti-cd11c fluorescence (red and top inset). 3-D colocalization (yellow and bottom inset) of the two channels predominantly at the leading edge and actin foci. (C) Proximity of podosomes to the substratum as shown by IRM (top inset). Colocalization (yellow and bottom inset) of phalloidin-FITC with the inverted IRM signal shows leading edge and podosome-containing regions in close proximity to the substratum. (D) Podosomes show a high correlation of integrin colocalization (blue, bottom inset in B) and substratum proximity (red, bottom inset in C) as indicated by the white signal of the majority of podosomes. Bars, 10 μm. (E and F) 3-D reconstruction of the boxed region in D shows that F-actin (green) is present in large (arrow) and small (arrowhead) podosomes, and that both are in close proximity to the substratum as shown by IRM (red) and contain integrin colocalization (yellow). Grid squares are 1 μm × 1 μm.

Mentions: Podosomes have classic features of a focal complex. Each actin-rich podosome is short-lived and punctate, contains actin cross-linking proteins (Marchisio et al., 1987; Babb et al., 1997), and when imaged by interference reflection microscopy (IRM), is in close apposition to the substratum (Izzard and Lochner, 1976; Tarone et al., 1985). Because podosomes are dynamic structures, we first verified when podosomes acquire key characteristics of a focal complex. Two markers for adhesions are colocalization with integrins (Marchisio et al., 1988) and close proximity to the substratum (Izzard and Lochner, 1976). Immunofluorescence micrographs show that cd11c (αX integrin) and F-actin colocalize at podosomes. When reconstructed in three dimensions, distinct subregions containing integrins are visible in the larger actin-containing structures (Fig. 1, E and F). Furthermore, actin- and integrin-containing structures colocalized with regions in close proximity to the substratum (Fig. 1, C–F). The coincidence of actin, integrin, and IRM signals suggests that the majority of podosomes at the leading edge are involved in adhesion to the substratum.


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)

Podosomes colocalize with integrins and are in close proximity to the substratum. (A) Maximum intensity projection of phalloidin-FITC fluorescence showing distribution of F-actin in an IC-21 macrophage. (B) Overlay of phalloidin-FITC (green) and anti-cd11c fluorescence (red and top inset). 3-D colocalization (yellow and bottom inset) of the two channels predominantly at the leading edge and actin foci. (C) Proximity of podosomes to the substratum as shown by IRM (top inset). Colocalization (yellow and bottom inset) of phalloidin-FITC with the inverted IRM signal shows leading edge and podosome-containing regions in close proximity to the substratum. (D) Podosomes show a high correlation of integrin colocalization (blue, bottom inset in B) and substratum proximity (red, bottom inset in C) as indicated by the white signal of the majority of podosomes. Bars, 10 μm. (E and F) 3-D reconstruction of the boxed region in D shows that F-actin (green) is present in large (arrow) and small (arrowhead) podosomes, and that both are in close proximity to the substratum as shown by IRM (red) and contain integrin colocalization (yellow). Grid squares are 1 μm × 1 μm.
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fig1: Podosomes colocalize with integrins and are in close proximity to the substratum. (A) Maximum intensity projection of phalloidin-FITC fluorescence showing distribution of F-actin in an IC-21 macrophage. (B) Overlay of phalloidin-FITC (green) and anti-cd11c fluorescence (red and top inset). 3-D colocalization (yellow and bottom inset) of the two channels predominantly at the leading edge and actin foci. (C) Proximity of podosomes to the substratum as shown by IRM (top inset). Colocalization (yellow and bottom inset) of phalloidin-FITC with the inverted IRM signal shows leading edge and podosome-containing regions in close proximity to the substratum. (D) Podosomes show a high correlation of integrin colocalization (blue, bottom inset in B) and substratum proximity (red, bottom inset in C) as indicated by the white signal of the majority of podosomes. Bars, 10 μm. (E and F) 3-D reconstruction of the boxed region in D shows that F-actin (green) is present in large (arrow) and small (arrowhead) podosomes, and that both are in close proximity to the substratum as shown by IRM (red) and contain integrin colocalization (yellow). Grid squares are 1 μm × 1 μm.
Mentions: Podosomes have classic features of a focal complex. Each actin-rich podosome is short-lived and punctate, contains actin cross-linking proteins (Marchisio et al., 1987; Babb et al., 1997), and when imaged by interference reflection microscopy (IRM), is in close apposition to the substratum (Izzard and Lochner, 1976; Tarone et al., 1985). Because podosomes are dynamic structures, we first verified when podosomes acquire key characteristics of a focal complex. Two markers for adhesions are colocalization with integrins (Marchisio et al., 1988) and close proximity to the substratum (Izzard and Lochner, 1976). Immunofluorescence micrographs show that cd11c (αX integrin) and F-actin colocalize at podosomes. When reconstructed in three dimensions, distinct subregions containing integrins are visible in the larger actin-containing structures (Fig. 1, E and F). Furthermore, actin- and integrin-containing structures colocalized with regions in close proximity to the substratum (Fig. 1, C–F). The coincidence of actin, integrin, and IRM signals suggests that the majority of podosomes at the leading edge are involved in adhesion to the substratum.

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