Macrophage podosomes assemble at the leading lamella by growth and fragmentation - PubMed (original) (raw)
Macrophage podosomes assemble at the leading lamella by growth and fragmentation
James G Evans et al. J Cell Biol. 2003.
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.
Figures
Figure 1.
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.
Figure 2.
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).
Figure 3.
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.
Figure 4.
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).
Figure 5.
A model for podosome assembly. Podosome assembly and disassembly takes place within an adhesion-rich zone. At the leading edge of the lamella, a podosome assembles de novo and disassembles in a microtubule-independent process. Podosome lifetime can be extended in a microtubule-dependent process by fission into daughter podosomes, fusion with another podosome, or growth into a PCP and fission into a podosome cluster. The assembly of daughter podosomes keeps pace with the leading lamella, whereas the mother podosome remains stationary until disassembling at the rear of the adhesion zone.
References
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