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Fig. 5. Optical highlighter FPs in action imaged with laser scanning confocal microscopy. (A-C) Photoactivation of mPA-GFP-actin-C-7 in opossum kidney (OK cell line) epithelial cells. (A) Circular region of interest selected with an Olympus FV1000 tornado scanner is illuminated at 405 nm for 5 seconds, t=0 minutes. (B) The photoactivated actin chimera first translocates to the ruffles at the cellular margins as fluorescence intensity decreases in the activated region, t=5 minutes. (C) Ruffles, cytoplasmic actin pools and the filamentous actin network gain more intensity at t=60 minutes. (D-F) Tracking of mitochondria labeled with tdEos-mito-N-7 in rabbit kidney (RK-13 cell line) epithelial cells. (D) Photoconversion of a single mitochondrion (red) in a selected region at 405 nm illumination, t=0 minutes. (E) Close approach of a non-converted (green) mitochondrion (arrow), t=10 minutes. (F) Cargo exchange between mitochondria (arrow), t=20 minutes. (G-I) Examination of lamellipodia with Dendra2-actin-C-7 in OK cells. (G) Photoconversion (red) of the selected region (box) with a 405 nm laser, t=0 minutes. (H) The photoconverted channel illustrates podosome formation by photoconverted actin and changes in leading edges, t=20 minutes. (I) Photoconverted lamellipod retracts amid increased podosome formation and generation of a new leading edge, t=45 minutes. (J-L) Photoswitching of the actin cytoskeleton with Dronpa-actin-C-7 in rat thoracic aorta (A7r5 cell line) myoblasts. (J) Actin network imaged with a 488-nm laser, t=0 minutes. (K) After completely photoswitching the labeled actin `off' at 488 nm, the region spelling FV10 was activated with a 405 nm laser, t=3 minutes. (L) FV10 region photobleached while imaging the actin network at 488 nm.
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