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Fig. 4. The level of microtubule acetylation in mature osteoclasts correlates with stabilisation of podosomes into belts. (A,B) Macrophages and immature osteoclasts, differentiated for 6 days in the presence of RANK-L + M-CSF, with podosome rings, presented a low level of acetylated tubulin. (C) Mature osteoclasts differentiated for 8 days in the presence of RANK-L + M-CSF presented specific accumulations of acetylated microtubules just behind the podosome belt (arrowhead). (D) This specific accumulation of acetylated microtubules during the transition period between podosome rings and belt, the last step of podosome patterning, was confirmed by western blot analysis on osteoclast populations differentiated for 6 (D6) or 8 (D8) days. Standard protein markers are indicated in kDa. (E,F) Rho inhibition accelerates the podosome ring to belt transition in immature osteoclasts. Spleen leukocytes were differentiated in the presence of RANKL and M-CSF for 6 days, and then treated or not with TAT-GFP or TAT-C3 for 6 hours before being fixed, and stained for F-actin with Phalloidin-RITC. A general overview of podosome organisation in osteoclasts is presented (E). At D6, osteoclasts in untreated or TAT-GFP treated cultures exhibited mostly podosome clusters or rings (arrowheads) or rare podosome belt (arrows). In contrast, in TAT-C3 treated osteoclasts, podosome belts were seen. This was quantified in osteoclasts containing more than three nuclei by counting 600 osteoclasts per condition (F). At this stage of differentiation, untreated osteoclasts or those treated for 6 hours with TAT-GFP (0.5 µM) exhibited a majority of podosomes arranged in rings or clusters, whereas TAT-C3 (0.5 µM) induced the formation of belts at the cell periphery as usually found in more mature osteoclasts. (G) Osteoclasts at 3, 5 and 7 days of differentiation were lysed to perform an affinity precipitation assay with GST-RBD fusion proteins. The amount of Rho in total cell lysates (Total Rho) and in the precipitated fraction (RhoGTP) was determined by western blotting using the 26C4 monoclonal anti-Rho antibody. The ratio of RhoGTP/total Rho was estimated by densitometry analysis. Bar 20 µm.
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