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. 2013 Dec 9;8(12):e77956. doi: 10.1371/journal.pone.0077956

Figure 5. Specific dynamin photoinactivation led to the rapid invadosome disorganization.

Figure 5

A) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-KR and GFP-paxillin. Dyn2-KR is functionnal while localized properly and rescued invadosome formation. KillerRed 45 s light irradiation is followed by the rapid dissociation of invadosome ring (dashed line) stained by GFP-paxillin also localized in focal adhesions (red arrows). B) Extracted images from time serie (min∶s) from representative observations of SKO-v-Src-MEFs expressing KillerRed (KR) alone and GFP-paxillin. Non-localized ROS production after light irradiation of KillerRed alone does not destabilize adhesions structures. C) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-pTRFP (same excitation/emission spectrum but much more photostable than KillerRed) and GFP-paxillin. Light irradiation without ROS production is not sufficient to dissociate invadosome structures. D) Extracted images from time serie (min∶s) from representative observations of SKO-v-Src-MEFs expressing GFP-paxillin and dyn2-KR. In presence of endogenous dynamin, photoinactivation of dyn2-KR has no effect on invadosome organization showing the localized and specificity of ROS produced by the photosensitizer on the proteins fused to it. E) Extracted images from time serie (min∶s) from representative observations of DKO-v-Src-MEFs expressing dyn2-GFP and dyn2-KR. ROS production at the level of the GTPase has no effect on dyn2-GFP stability visualizing and confirming the previous experiment. F) Distribution of the percentage of cells where invadosome structures is disorganized×min after light irradiation. The >17,5 min category corresponds to experiments where invadosome presented a life-span superior to 17,5 min (distributed between 20 and 40 min, depending of the duration of each movies) and pulled altogether. 16 to 56 cells per conditions were monitored. Scale bar = 2 µm (A, E), 4 µm (B, C, D).