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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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Podosome clusters arise from PCPs. (A) Frames from a 2-D wide-field time-lapse experiment showing L-fimbrin–EGFP fluorescence at the leading edge of an IC-21 macrophage. An amorphous area of fluorescence (PCP, dashed line and arrowhead) precedes a cluster of individual podosomes (arrows). Bar, 10 μm; 1 μm (inset). (B) In a 4-D experiment, SFP rendering of β-actin–EYFP fluorescence at the leading edge of an IC-21 macrophage shows a PCP (arrowhead) followed a few minutes later by a cluster of smaller, discrete podosomes (arrows). Deconvolved data were rendered with the SFP algorithm in which objects, illuminated from above, cast a shadow aiding visual perception of 3-D structures. Maximum intensity projections of the same data are shown in the insets (see Video 2) Bars, 3 μm. (C) XY (left) and XZ (middle) views of isosurface reconstructions showing that a podosome cluster (red) formed from a PCP (green). Panels on the right show color-coded time points (green the earliest, red the latest). Bar, 1 μm. (D) Podosome cluster formation visualized by maximum intensity projection of each 3-D time point and tracing the high intensity structures over time. In the kymograph (gray, offset in main panels, side view left panel inset), the PCP (green) is seen to assemble (long single trace) and then simultaneously form several new podosomes (branches, blue and red structures). Side projection of the kymograph (inset) follows the direction of lamellar extension (arrow). Grid squares are 2 μm (x, y) and 1 min (t).
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fig2: Podosome clusters arise from PCPs. (A) Frames from a 2-D wide-field time-lapse experiment showing L-fimbrin–EGFP fluorescence at the leading edge of an IC-21 macrophage. An amorphous area of fluorescence (PCP, dashed line and arrowhead) precedes a cluster of individual podosomes (arrows). Bar, 10 μm; 1 μm (inset). (B) In a 4-D experiment, SFP rendering of β-actin–EYFP fluorescence at the leading edge of an IC-21 macrophage shows a PCP (arrowhead) followed a few minutes later by a cluster of smaller, discrete podosomes (arrows). Deconvolved data were rendered with the SFP algorithm in which objects, illuminated from above, cast a shadow aiding visual perception of 3-D structures. Maximum intensity projections of the same data are shown in the insets (see Video 2) Bars, 3 μm. (C) XY (left) and XZ (middle) views of isosurface reconstructions showing that a podosome cluster (red) formed from a PCP (green). Panels on the right show color-coded time points (green the earliest, red the latest). Bar, 1 μm. (D) Podosome cluster formation visualized by maximum intensity projection of each 3-D time point and tracing the high intensity structures over time. In the kymograph (gray, offset in main panels, side view left panel inset), the PCP (green) is seen to assemble (long single trace) and then simultaneously form several new podosomes (branches, blue and red structures). Side projection of the kymograph (inset) follows the direction of lamellar extension (arrow). Grid squares are 2 μm (x, y) and 1 min (t).

Mentions: In live cells observed with phase optics, podosomes appear as small, phase-dense structures that continuously form at the periphery of the macrophage lamella (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200212037/DC1). When marked by GFP-tagged fimbrin or actin, podosomes appear and disappear within a few minutes at the leading lamella, confirming previous observations (Kanehisa et al., 1990). However, in addition to the rapid turnover of many podosomes, time-lapse microscopy showed the transient presence of a large amorphous fimbrin- and actin-containing structure immediately preceding the appearance of a cluster of four to six podosomes (Fig. 2 A). To identify the function of this structure more clearly, we imaged the entire axial volume of the macrophage lamella by three-dimensional (3-D) time-lapse (4-D) microscopy (Fig. 2 B and Video 2). In 3-D reconstructions, the volume of the large amorphous structure was 5–10-fold larger than individual podosomes (5–10 fL vs. 0.5–2 fL) and preceded podosomes by 1–2 min (Fig. 2 B and Video 3). The axial height of PCPs and podosomes was ∼1–2 μm (Fig. 2 C), a length sufficient to span the distance between dorsal and ventral plasma membranes in the lamella as measured previously by EM (Trotter, 1981; Kato and Akisaka, 1994). We termed the structure a podosome cluster precursor because it appeared to generate several new juxtaposed podosomes.


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)

Podosome clusters arise from PCPs. (A) Frames from a 2-D wide-field time-lapse experiment showing L-fimbrin–EGFP fluorescence at the leading edge of an IC-21 macrophage. An amorphous area of fluorescence (PCP, dashed line and arrowhead) precedes a cluster of individual podosomes (arrows). Bar, 10 μm; 1 μm (inset). (B) In a 4-D experiment, SFP rendering of β-actin–EYFP fluorescence at the leading edge of an IC-21 macrophage shows a PCP (arrowhead) followed a few minutes later by a cluster of smaller, discrete podosomes (arrows). Deconvolved data were rendered with the SFP algorithm in which objects, illuminated from above, cast a shadow aiding visual perception of 3-D structures. Maximum intensity projections of the same data are shown in the insets (see Video 2) Bars, 3 μm. (C) XY (left) and XZ (middle) views of isosurface reconstructions showing that a podosome cluster (red) formed from a PCP (green). Panels on the right show color-coded time points (green the earliest, red the latest). Bar, 1 μm. (D) Podosome cluster formation visualized by maximum intensity projection of each 3-D time point and tracing the high intensity structures over time. In the kymograph (gray, offset in main panels, side view left panel inset), the PCP (green) is seen to assemble (long single trace) and then simultaneously form several new podosomes (branches, blue and red structures). Side projection of the kymograph (inset) follows the direction of lamellar extension (arrow). Grid squares are 2 μm (x, y) and 1 min (t).
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fig2: Podosome clusters arise from PCPs. (A) Frames from a 2-D wide-field time-lapse experiment showing L-fimbrin–EGFP fluorescence at the leading edge of an IC-21 macrophage. An amorphous area of fluorescence (PCP, dashed line and arrowhead) precedes a cluster of individual podosomes (arrows). Bar, 10 μm; 1 μm (inset). (B) In a 4-D experiment, SFP rendering of β-actin–EYFP fluorescence at the leading edge of an IC-21 macrophage shows a PCP (arrowhead) followed a few minutes later by a cluster of smaller, discrete podosomes (arrows). Deconvolved data were rendered with the SFP algorithm in which objects, illuminated from above, cast a shadow aiding visual perception of 3-D structures. Maximum intensity projections of the same data are shown in the insets (see Video 2) Bars, 3 μm. (C) XY (left) and XZ (middle) views of isosurface reconstructions showing that a podosome cluster (red) formed from a PCP (green). Panels on the right show color-coded time points (green the earliest, red the latest). Bar, 1 μm. (D) Podosome cluster formation visualized by maximum intensity projection of each 3-D time point and tracing the high intensity structures over time. In the kymograph (gray, offset in main panels, side view left panel inset), the PCP (green) is seen to assemble (long single trace) and then simultaneously form several new podosomes (branches, blue and red structures). Side projection of the kymograph (inset) follows the direction of lamellar extension (arrow). Grid squares are 2 μm (x, y) and 1 min (t).
Mentions: In live cells observed with phase optics, podosomes appear as small, phase-dense structures that continuously form at the periphery of the macrophage lamella (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200212037/DC1). When marked by GFP-tagged fimbrin or actin, podosomes appear and disappear within a few minutes at the leading lamella, confirming previous observations (Kanehisa et al., 1990). However, in addition to the rapid turnover of many podosomes, time-lapse microscopy showed the transient presence of a large amorphous fimbrin- and actin-containing structure immediately preceding the appearance of a cluster of four to six podosomes (Fig. 2 A). To identify the function of this structure more clearly, we imaged the entire axial volume of the macrophage lamella by three-dimensional (3-D) time-lapse (4-D) microscopy (Fig. 2 B and Video 2). In 3-D reconstructions, the volume of the large amorphous structure was 5–10-fold larger than individual podosomes (5–10 fL vs. 0.5–2 fL) and preceded podosomes by 1–2 min (Fig. 2 B and Video 3). The axial height of PCPs and podosomes was ∼1–2 μm (Fig. 2 C), a length sufficient to span the distance between dorsal and ventral plasma membranes in the lamella as measured previously by EM (Trotter, 1981; Kato and Akisaka, 1994). We termed the structure a podosome cluster precursor because it appeared to generate several new juxtaposed podosomes.

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