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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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Actin and fimbrin dynamics at the leading edge. (A) 2-D time-lapse deconvolution confocal microscopy of β-actin–ECFP (green) and L-fimbrin–EYFP (red) recorded at 15-s intervals for 30 min. Colocalized signals are shown in yellow. Insets show the boxed region at higher magnification at t = 0 (left) and 30 min later (right). Bar, 10 μm. (B) Kymographs from the boxed region (A) shows that actin (left, green) and fimbrin (right, red) have similar lateral distribution over time producing short-lived traces (arrowhead) and longer-lived branching traces (arrow). A 2-D image of the last time point forms the floor of the kymograph box. The boxed region is 15 μm × 20 μm × 30 min.
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fig3: Actin and fimbrin dynamics at the leading edge. (A) 2-D time-lapse deconvolution confocal microscopy of β-actin–ECFP (green) and L-fimbrin–EYFP (red) recorded at 15-s intervals for 30 min. Colocalized signals are shown in yellow. Insets show the boxed region at higher magnification at t = 0 (left) and 30 min later (right). Bar, 10 μm. (B) Kymographs from the boxed region (A) shows that actin (left, green) and fimbrin (right, red) have similar lateral distribution over time producing short-lived traces (arrowhead) and longer-lived branching traces (arrow). A 2-D image of the last time point forms the floor of the kymograph box. The boxed region is 15 μm × 20 μm × 30 min.

Mentions: Analysis of a single podosome cluster implicated growth and fragmentation as a mechanism for the generation of new podosomes. To study the assembly mechanism in more detail, we developed algorithms for measuring key parameters of podosome dynamics; the lifetime and fission frequency of all podosomes in an imaged volume (x, y, t) of the cell. Because it is implicated in organizing actin in podosomes (Babb et al., 1997), we also analyzed the dynamics of the actin cross-linking protein fimbrin. Analysis of time-lapse movies (Fig. 3 A) showed that β-actin–ECFP and L-fimbrin–EYFP colocalized to the core of each podosome. In kymographs, fimbrin revealed a pattern of dynamics that was coincident in space and time to that of actin (Fig. 3 B). Kymograph traces from actin and fimbrin showed both short-lived and longer-lived branching structures. We were unable to detect any temporal difference in actin and fimbrin localization during assembly or turnover of podosomes. These observations suggest that actin and fimbrin are closely coupled dynamically in 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)

Actin and fimbrin dynamics at the leading edge. (A) 2-D time-lapse deconvolution confocal microscopy of β-actin–ECFP (green) and L-fimbrin–EYFP (red) recorded at 15-s intervals for 30 min. Colocalized signals are shown in yellow. Insets show the boxed region at higher magnification at t = 0 (left) and 30 min later (right). Bar, 10 μm. (B) Kymographs from the boxed region (A) shows that actin (left, green) and fimbrin (right, red) have similar lateral distribution over time producing short-lived traces (arrowhead) and longer-lived branching traces (arrow). A 2-D image of the last time point forms the floor of the kymograph box. The boxed region is 15 μm × 20 μm × 30 min.
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Related In: Results  -  Collection

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fig3: Actin and fimbrin dynamics at the leading edge. (A) 2-D time-lapse deconvolution confocal microscopy of β-actin–ECFP (green) and L-fimbrin–EYFP (red) recorded at 15-s intervals for 30 min. Colocalized signals are shown in yellow. Insets show the boxed region at higher magnification at t = 0 (left) and 30 min later (right). Bar, 10 μm. (B) Kymographs from the boxed region (A) shows that actin (left, green) and fimbrin (right, red) have similar lateral distribution over time producing short-lived traces (arrowhead) and longer-lived branching traces (arrow). A 2-D image of the last time point forms the floor of the kymograph box. The boxed region is 15 μm × 20 μm × 30 min.
Mentions: Analysis of a single podosome cluster implicated growth and fragmentation as a mechanism for the generation of new podosomes. To study the assembly mechanism in more detail, we developed algorithms for measuring key parameters of podosome dynamics; the lifetime and fission frequency of all podosomes in an imaged volume (x, y, t) of the cell. Because it is implicated in organizing actin in podosomes (Babb et al., 1997), we also analyzed the dynamics of the actin cross-linking protein fimbrin. Analysis of time-lapse movies (Fig. 3 A) showed that β-actin–ECFP and L-fimbrin–EYFP colocalized to the core of each podosome. In kymographs, fimbrin revealed a pattern of dynamics that was coincident in space and time to that of actin (Fig. 3 B). Kymograph traces from actin and fimbrin showed both short-lived and longer-lived branching structures. We were unable to detect any temporal difference in actin and fimbrin localization during assembly or turnover of podosomes. These observations suggest that actin and fimbrin are closely coupled dynamically in 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