JCS03271 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Podosome fission and fusion. Frames from movies of osteoclasts expressing GFP-paxillin and Cherry-actin. Clustered podosomes showing podosome fission (A) and fusion (B). Numbers indicate time after initiation of the time-lapse recording, in seconds. Bars, 1 μm.

• Supplemental Figure 2 -

  Fig. S2. Src affects podosome number. (A) Western blot for Src in RAW-derived osteoclasts (control), Src251 osteoclasts, and SrcY527F osteoclasts. (B) Number of podosomes per mm² in cluster podosomes from non-polarized control osteoclasts (six cells, 163±154 podosomes), SZL structure podosomes from polarized control osteoclasts (six cells, 62±24 podosomes), Src 251 osteoclasts (nine cells, 455±396 podosomes), SrcY527F osteoclasts (six cells, 167±69 podosomes).

• Supplemental Figure 3 -

  Fig. S3. Src localization in osteoclasts. Osteoclasts derived from RAW cells expressing GFP-actin (control) or RAW cells co-expressing either GFP-actin and SrcY527F (srcY527F) or GFP-actin and Src251 (Src251), were fixed and double-stained for Src and actin. (A) Notice limited co-localization at the SZL structures in control and SrcY527F cells, and very high co-localization in podosomes of Src251 cells. Bar, 10 μm. (B) confocal microscopy X-Z images showing co-localization of the two proteins throughout the actin core. Bars, 1 μm.

• Supplemental Figure 4 -

  Fig. S4. Localization of phospho-cortactin. RAW-derived osteoclasts were transfected with GFP-paxillin, fixed, and stained for cortactin and cor pY421. Note the co-localization between phosphocortactin and paxillin. Bar, 10 μm.

• Supplemental Figure 5 -

  Fig. S5. Differential cortactin phosphorylation during osteoclast polarization. Primary murine osteoclast-like cells were prepared as previously described (Chiusaroli et al., 2004), fixed, and immunoassayed for actin, cortactin and cortactin pY421(A and B). (A) Notice co-localization of the two proteins in podosomes of SZL structures (arrow) and scattered podosomes (arrowhead). (B) Cortactin/cortactin pY421 ratio shows cortactin phosphorylation in scattered but not ring-associated podosomes. Bar, 10 μm.

• Movie 1 -

  Movie 1. Podosomes in non-polarized osteoclasts. GFP-actin, shown in inverted contrast for better visualization. Black dots represent a fluorescent podosome. Notice the increase in actin dynamics upon cluster-ring transition. Images were acquired every 15 seconds Total time: 30 minutes. Display rate: 5 frames/second. Objective lens: ×100/1.3.

• Movie 2 -

  Movie 2. Podosomes in polarized osteoclasts. GFP-actin, shown in inverted contrast for better visualization. Black dots represent fluorescent podosomes. Notice highly dynamic podosomes giving rise to stable SZL structures at the cell periphery. Images were acquired every 15 seconds. Total time: 10 minutes. Display rate: 5 frames/second. Objective lens: ×100/1.3.

• Movie 3 -

  Movie 3. Temporal ratio movie of a polarized control cell. GFP-actin images were processed for this temporal ratio movie. Images were acquired every 15 seconds. Total time: 15 minutes. Display rate: 5 frames/second. Objective lens: ×100/1.3. See also Fig. 4A.

• Movie 4 -

  Movie 4. Temporal ratio movie of a cell overexpressing srcY527F. GFP-actin pictures were processed for this temporal ratio movie. Notice the disrupted SZL rings at the cell periphery, and the ectopic podosomes scattered throughout the ventral membrane. Images were acquired every 30 seconds. Total time: 15 minutes. Display rate: 5 frames/second. Objective lens: ×100/1.3. See also Fig. 4A.

• Movie 5 -

  Movie 5. Temporal ratio movie of a cell overexpressing src251. GFP-actin pictures were processed for a temporal ratio movie. Notice large podosome clusters throughout the ventral membrane. Images were acquired every 30 seconds. Total time: 30 minutes. Display rate: 5 frames/second. Objective lens: ×100/1.3. See also Fig. 4A.