Dynamics of the sealing zone in cultured osteoclasts

Abstract

Bone resorption by osteoclasts (OCs) depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in OCs cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity. Double labeling of cells for actin and tubulin indicated that microtubules (MTs) are mostly confined by the inner aspect of the stable SZL-associated actin belt. However, in unstable regions of the SZL, MTs tend to extend radially, across the SZL, toward the cell edge. Disruption of MTs by nocodazole induces SZ disassembly, without affecting individual podosome stability. Inspection of the MT network indicates that it is enriched along stable SZL regions, while bypassing disorganized regions. These results suggest that the SZL is stabilized by MTs flanking its inner aspect, while disruption or misalignment of MTs leads to SZL destabilization. We further demonstrate that the MT-associated protein dynamin2 is involved in the regulation of SZL stability, and dynamin2 knockdown or inactivation cause SZL destabilization.

Keywords: RAW cells; dynamin2; osteoclasts; podosomes; sealing zone-like podosome belt.

Figure 1

Assessment of SZL dynamics. (a) Actin‐GFP‐tagged OCs were fluorescently imaged at a rate of one frame every 30 s. Images of the SZL‐associated actin were outlined manually, frame‐by‐frame, in four movies, one presented here, and linearized (start point indicated by the yellow arrow). The path was then divided into 50 pixel‐long segments; for each segment, four properties were quantified: (1) Average intensity (in 5 pixel‐wide strips taken along the segment); (2) Curvature (inverse radius of fitted circle, defined as positive for the convex curve with respect to the cell center); (3) velocity (translocation of the SZL segment center from one timeframe to the next (displacement away from the cell center was defined as positive), and (4) pixel‐by‐pixel variance within the strip area (contiguous belts yield low variance, whereas clusters of individual podosomes yield high variance). Variance is calculated as sqrt[<(I(x,y) − I _avg)²>]/I _avg; I _avg = <I(x,y)>, where I(x,y) is image intensity at pixel (x,y), and <> marks the averaged variance in the region of interest. The values of these properties are color‐coded, and displayed as four slightly shifted belt path outlines. Distances from the start of the path are marked in white numbers along the path. (b) Presentation of the color‐coded values along the “straightened” SZL path, stacked as a function of time to display as kymographs. Each of the four horizontal strips represents the quantified properties along the path. The kymographs illustrate regions with a strong correlation between high belt concave curvature and fast belt velocity. These regions also correlate with low actin intensity and high variance, and are usually flanked by high actin intensity regions with low variance; see black arrows. Scale bar: 25 µm.To quantify this data, we calculated Pearson correlation coefficients between the 6 pairs of the 4 measured curves, averaged on the whole path and over 40 time frames for 4 movies. The correlation coefficients are averaged on the entire SZL path. Values obtained for selected regions yield higher correlations; Curvature‐Velocity: Cor1 = −0.6 ± 0.3; Curvature‐Intensity: Cor2 = 0.4 ± 0.2; Velocity‐Intensity: Cor3 = −0.3 ± 0.1; Curvature‐Variance: Cor4 = 0.3 ± 0.2; Velocity‐Variance: Cor5 = 0.2 ± 0.1; Intensity‐Variance: Cor6 = −0.2 ± 0.1.

Figure 2

Quantitative analysis of SZL features. (a) Segments (20 μm‐long) along the movies of SZL paths were outlined by polygons, and total actin intensity was quantified as a function of time. Total intensities include both distinct podosomes and “cloud” actin contributions. About 5 min‐long cycles of increasing and decreasing intensity are shown in three such regions for the cell shown in Figure 1; see red, yellow, and green polygons in (c). Lack of synchronization between these cyclic behaviors in the various regions, visually apparent in all movies we acquired, suggests local, rather than global, regulation of SZL stability. (b) Autocorrelations (averaged over more than four assembly disassembly cycles) for the three curves shown in Figure 2a, indicating that they are essentially identical, and implying a lack of long‐term instability in specific regions along the SZL. Kolmogorov‐Smirnov comparison p‐value = 0.996. Although the curve is not strictly a single exponent, an exponential fit for the fast region yields τ = 1 $\pm$ 0.2 min (n = 10).

Figure 3

Nocodazole treatment inhibits podosome compaction in the SZL. Time‐lapse movies of actin‐GFP‐tagged OCs were acquired at a rate of one frame every 30 s. Following 10 min of recording, 5 µM of nocodazole was added to the cells. (a) SZL recorded prior to nocodazole treatment. (b, c) SZL recorded 1 and 2 min post‐nocodazole treatment. (d) Time‐dependence analysis of podosome distances from the SZL prior to and during nocodazole treatment. Green dots correspond to podosome centers; the vertical axis indicates distance from the SZL; the horizontal axis indicates time. The yellow curve represents the time dependence of twice the average podosome distance (enveloping the green dots for visualization). The white arrow indicates the time of nocodazole addition. Average podosome dispersion time measured at 25 μm distance is 1 $\pm$ 0.3 min (n = 4). The distance between the dispersing podosomes (evaluated by the average of minimal distances to closest neighbor, or the average of minimal distances to their five closest neighbors) does not change significantly, as their numbers increase, and they occupy a wider cell area. Scale bar: 40 µm.

Figure 4

3D view of MTs and actin in a mature OC. (a) Focal series of a fully differentiated OC, stained for MT and actin. The 3D series was acquired and subjected to deconvolution and 3D reconstruction by means of Imaris software. (b, c) Two optical sections taken at near‐slide level, showing actin and MTs, respectively. MT ends reaching stable SZL regions (Arrows 1‐2) can be observed. (d) MTs located 3 μm above the slide surface. MTs run across the unstable region of the SZL toward the cell periphery (Arrow 3). (e) These features are also displayed by the height‐coded MT projection image (red indicates high; blue/green indicates low).

Figure 5

MTs, actin and dynamin2 colocalize. Super‐resolution images taken from OCs triple‐labeled for MT, actin and dynamin2. (a) MTs in red, with dynamin2 in yellow. (b) Zoom‐in view, showing that most dynamin2 dots lie near MTs. (c) Actin height color scale. Podosomes (indicated by the arrows) display higher actin loci (yellow). (d) Double labeling for actin (in red) and dynamin2 (in yellow), indicating an abundance of dynamin2 near podosomes. 20% of dynamin2 dots are at a 1 μm distance from podosome centers.

Figure 6

Treatment of OCs with the dynamin2 inhibitor Dyngo‐4a inhibits podosome compaction in the SZL. A time‐lapse movie of actin‐GFP‐expressing OCs was recorded at 30 s intervals. Following 10 min of recording, 5 µM of Dyngo‐4a was added to the samples. (a) Image taken prior to Dyngo‐4a treatment. (b, c) Images taken 2 and 4 min after Dyngo‐4a treatment. (d) Time‐dependence analysis of podosome distances from the SZL prior to and during Dyngo treatment. Green dots indicate podosome centers. Vertical axis—distance from the SZL; horizontal axis corresponds to time. The yellow curve shows the time dependence of twice the average podosome distance (enveloping the green dots for visualization). White arrow indicate time of Dyngo‐4a addition. Average podosome dispersion time measured at 25 μm distance is 1 $\pm$ 0.4 min (n = 6). The distance between the dispersing podosomes (evaluated by the average of minimal distances to closest neighbor, or the average of minimal distances to five closest neighbors) does not change significantly, as their number increases and they occupy a wider cell area. Scale bar: 25 µm.