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First published online 14 November 2006
doi: 10.1242/jcs.03271

Journal of Cell Science 119, 4878-4888 (2006)
Published by The Company of Biologists 2006
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Involvement of the Src-cortactin pathway in podosome formation and turnover during polarization of cultured osteoclasts

Chen Luxenburg1, J. Thomas Parsons2, Lia Addadi3 and Benjamin Geiger1,*

1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
2 Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA
3 Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel


Figure 1
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  		Fig. 1. Dynamic development of podosome organization during osteoclast polarization. RAW cells, either stably expressing GFP-actin or transiently transfected with YFP-paxillin, were induced to differentiate into osteoclasts and were recorded at 15-second intervals by time-lapse microscopy. (A) Temporal ratio images of GFP-actin. Four distinct, consecutive stages of podosome organization are shown: Individual podosomes (ind pod/cluster) (see arrow); clusters; rings; and sealing-zone-like (SZL) structures. Temporal ratio images indicate new podosomes (~1-minute old) in blue, podosomes that disappeared within the same minute of recording, in red, and more stable podosomes in yellow (see color legend in Figure). Actin reorganization in individual podosomes and clusters is slower than in ring and SZL structures. Note that clustered podosomes are less dynamic than ring podosomes within the same cell (ring, marked with arrowhead). (B) The quantitative data represent the average podosome life span (ALS) ± s.d., based on GFP-actin movies of five cells for every structure, obtained in three independent experiments. A total of 240 podosomes were examined for clusters and SZL structures; 220 podosomes were examined for rings. Statistical analysis based on the Student's t-test showed significant differences between cluster and ring structures (P<5 x10-41), and cluster and SZL structures (P<5 x10-41). (C) Temporal ratio image of YFP-paxillin. Paxillin ring domains in individual podosomes and clusters appeared less dynamic than in rings. Note the transition from an individual podosome to a cluster (ind pod/cluster 1' and 4', arrow). The SZL structure is a stable, continuous structure. The apparent sliding of the SZL structure was evident in the 10' temporal ratio image. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Podosome fusion, fission and sliding. (A) Podosome fusion within a cluster. Frames from a GFP-actin movie show two neighboring podosomes (0-60", arrows) that approach each other and fuse (75-105", arrowheads). (B) Podosome fission within a cluster. Frames from a GFP-actin movie show podosome splitting (0-30", arrows), creating two daughter podosomes (45"-75", arrowheads). (C) Apparent sliding motion of a podosome within a cluster. Frames from a GFP-actin movie are shown. Podosomes recorded at three time points are marked with different artificial colors (arrows indicate the original localization of the podosome). Time-lapse movies of cells expressing (D) GFP-actin or (E) YFP-paxillin analyzed by temporal autocorrelation techniques. The green line represents the correlation decay time constant (1/e). Five cells were analyzed for every podosome pattern. The blue line represents cluster podosomes and the red line represents SZL structure podosomes. (D) GFP-actin cluster podosomes decayed after 105 seconds; GFP-actin SZL structure decayed after 30 seconds. (E) YFP-paxillin in the cluster decayed after 195 seconds; YFP-paxillin in the SZL structure decayed after 225 seconds. Bars, 1 µm.

 

Figure 3
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  		Fig. 3. Changes in the levels of podosome-associated actin and paxillin during cluster-ring transition. RAW cells expressing GFP-actin or YFP-paxillin were induced to differentiate into osteoclasts, and recorded at 15-second intervals by time-lapse video microscopy. Frames are presented in an intensity spectrum scale. A negative time point indicates seconds before the transition. Time point 0 indicates the beginning of cluster-ring transition. (A) An increase in GFP-actin intensity (about tenfold) is evident during cluster-ring transition (0-105"). (B) A slight decrease in YFP-paxillin intensity is noted during cluster-ring transition (0-105"). Arrows indicate the ring. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Differential tyrosine phosphorylation of podosomes during osteoclast polarization. RAW cells were induced to differentiate, and then fixed and double-stained for actin and phosphotyrosine (PY). Actin and PY are shown in an intensity spectrum scale. Ratio images (PY/act ratio) demonstrate tyrosine phosphorylation of the entire actin domain of clustered podosomes (b) whereas in the SZL structure (a) tyrosine phosphorylation is predominantly associated with the plaque domain. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Effect of modulation of Src activity on podosome organization and dynamics. Temporal ratio imaging of GFP-actin, based on time-lapse microscopy of osteoclasts expressing GFP-actin (control); co-expressing GFP-actin and SrcY527F (SrcY527F); or co-expressing or GFP-actin and Src251 (Src251). Notice the elevated rate of podosome turnover and formation of ectopic podosomes in the SrcY527F-expressing cells, and the predominance of stationary podosomes in the Src251-expressing cells (0.5' and 10'). The calculated average podosome life span (± s.d.) is 52±25 seconds in SZL structures of SrcY527F cells, and 31±15 seconds in the ectopic podosomes of the same cells. The life span of the dynamic podosomes in Src251-expressing osteoclasts was 528±334 seconds. These data were based on measurements taken in five cells, with >200 podosomes for each cell type. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. Src catalytic activity affects podosome development. Osteoclasts derived from RAW cells expressing GFP-actin (control) or co-expressing GFP-actin and SrcY527F (SrcY527F), or GFP-actin and Src251 (Src251), were fixed and double-stained (A) for vinculin and actin, or (B) for Src pY418 and actin. (A) The vinculin/actin ratio indicates a typical double ring structure in control cells. Notice the poorly organized SZL structure in the SrcY527F cell and podosomes with incomplete ring domains, organized in clusters, in the Src251-expressing cells. (B) High levels of Src pY418 (src418) in the inner belt surround control and SrcY527F cells, and surround podosomes in Src251 cells. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Differential cortactin phosphorylation. (A) Control RAW osteoclasts, (B) cells co-expressing GFP-actin and SrcY527F, or (C) cells co-expressing GFP-actin and Src251 were fixed and triple-stained for actin (act), cortactin (cor) and phosphorylated cortactin (cor pY421). (A) Cortactin/actin ratio image highlights cortactin association with both SZL structure and clustered podosomes. Note the dramatic decrease in cortactin phosphorylation at the SZL structure (cor pY421/cor ratio image). (B) In SrcY527F-expressing cells, cortactin phosphorylation is moderately increased (cor pY421/cor ratio). (C) In Src251-expressing cells, cortactin is highly phosphorylated (cor pY421/cor ratio). Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Overexpression of cortactin mutants decreases cortactin phosphorylation in podosomes. RAW-derived osteoclasts were co-transfected with GFP-actin and corWT-flag or GFP-actin and cor3YF-flag. Cells were fixed and stained for the flag tag. (A) CorWT and (B) cor3YF localized to podosomes. (C) Cells were fixed and stained for cortactin and cortactin pY421. Five control cells, eight cells expressing corWT and eight cells expressing cor3YF were segmented and the fluorescence intensity of their podosomes was determined. In control cells, the average cortactin intensity calculated (±s.d.) was 61±27 (cortactin) and 432±132 (cortactin pY421). In cells overexpressing corWT, the average intensities calculated were 638±288 (cortactin) and 811±271 (cortactin pY421). In cells overexpressing cor3YF, the average cortactin intensity calculated (±s.d.) was 512±230 (cortactin) and 501±189 (cortactin pY421). Note the ten- or eightfold increase in cortactin intensity in cells expressing corWT or cor3YF, respectively, and only a 45% or 16% increase in cortactin phosphorylation in these cells. (D) The ratio of phosphocortactin to general cortactin intensities.

 

Figure 9
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  		Fig. 9. Cortactin phosphorylation affects podosome dynamics. (A) RAW-derived osteoclasts were transfected with GFP-actin and dsRED (control), with GFP-actin and wild-type cortactin (corWT), or with GFP-actin and a cortactin phosphorylation mutant (cor3YF). Temporal ratio images, based on time-lapse microscopy of GFP-actin, are shown. Notice the decrease in stable podosomes in cells overexpressing either WT or mutant cortactin (corWT, cor3YF). (B) The calculated average (±s.d.) life span of cluster podosomes was 169±107 seconds in control cells, 128±73 seconds in cells overexpressing WT cortactin, and 84±54 seconds in cells overexpressing cortactin 3YF. For control cells, a total of 159 podosomes from four cells in three independent experiments were analyzed; for cells expressing WT cortactin, seven cells from three experiments, with a total of 238 podosomes were examined; for cortactin 3YF, nine cells from four experiments, with a total of 402 podosomes, were examined. Statistical differences were calculated using the Student's t-test (control vs. corWT cells: P<5 x10-4; control vs. cor3YF, and corWT vs. cor3YF cells: P<5 x10-13). (C) RAW cells co-expressing GFP-actin and Src251 were transfected with dsRED (control); dsRED and WT cortactin (corWT); or dsRED and cor3YF. Temporal ratio images based on time-lapse microscopy of GFP-actin are shown. Notice the increase in podosome dynamics without the development of rings or SZL structures, in cells overexpressing corWT and cor3YF. Bars, 10 µm.

 

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© The Company of Biologists Ltd 2006