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Fig. 1. TAT-C3-mediated Rho inhibition in osteoclasts confers podosome belt resistance to microtubule depolymerisation through stabilisation of a subset of microtubules. Rho-inhibition partially blocks nocodazole-induced microtubule depolymerisation and podosome belt dissociation. Osteoclasts either untreated or treated for 5 hours in the presence of TAT-GFP (0.5 µM) or TAT-C3 (0.5 µM) were incubated in the presence of nocodazole (2 µM) for 50 minutes and then fixed and stained for actin (in red) and ß-tubulin (in green) before observation using a confocal microscope. (A) Kinetics of nocodazole-mediated podosome belts and microtubule disruption. In the presence of the control (TAT-GFP), nocodazole disrupted both microtubules and podosome belts in less than 30 minutes. In the presence of TAT-C3, podosomes belts were resistant to nocodazole treatment for more than 1 hour whereas subsets of microtubules were still observed. (B) TAT-C3 pretreatment had no effect on osteoclast cytoskeletons exhibiting a dense microtubule network and a podosome belt, whereas nocodazole induced complete microtubule dissociation together with podosome belt destabilisation and the subsequent formation of podosome rings (arrowheads) and clusters (open arrowheads) as previously described (Destaing et al., 2003). In contrast, TAT-C3 blocked the action of nocodazole since podosome belts were stabilised at the osteoclast periphery (arrows) and a subset of microtubules was maintained. A close-up of the area within the white insert is presented underneath each image. (C) Rho activation induces microtubule stabilisation in nocodazole-treated NIH3T3 cells. NIH3T3 cells were serum starved for 12 hours in the presence of TAT-GFP or TAT-C3 (0.5 µM) for the last 4 hours. Then cells were stimulated by serum addition for a further 2 hours before a 50-minute nocodazole (2 µM) treatment. Nocodazole-resistant microtubules were barely detectable when Rho was inactivated either in the absence of serum or in the presence of TAT-C3. In contrast, Rho activation by serum induced microtubule stabilisation in TAT-GFP control cells. A close-up of the area within the white insert is presented underneath each image. Bar, 10 µm (A, lower panels in B, C); 20 µm (upper panels in B).
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