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Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
^* Author for correspondence (e-mail: bochang{at}ust.hk )

View larger version (35K): [in a new window]   Fig. 3. Quantitative analysis of the distribution of cytochrome c and mitochondrial diameter change. (A,B) Magnified images of Mitotracker before (A) and after (B) mitochondria swelled. (C) The diameter of a mitochondrion was taken as the half width of its pixel profile based on a line-scan measurement of the Mitotracker image. (D) The distribution profiles of cytoc-GFP fluorescence intensity in a region containing only cytoplasm (left), or in a region containing both cytoplasm and mitochondria (right). The arrow marks the threshold that separates the fluorescence signal contributed by cytoc-GFP located in the mitochondria from that of the cytosolic cytoc-GFP.

View larger version (37K): [in a new window]   Fig. 1. Change of mitochondrial morphology during swelling. Mitochondria within a HeLa cell were stained by Mitotracker and imaged using a fluorescence microscope. (A) Under the normal condition, mitochondria appeared as filamentous structures. (B) After mitochondria were induced to swell by exposing the cell to a hypo-osmotic medium (5 mM potassium phosphate buffer, pH 7.4) for 1 minute, mitochondria were found to change into spherical shapes. (C) Calculated changes in volume and diameter when a mitochondrion swelled without stretching its surface. The length/diameter ratio was assumed to be five before swelling (Scheffler, 1999). Scale bar: 10 µm.

View larger version (100K): [in a new window]   Fig. 2. (A-F) Sample records from a time-series confocal measurements of cytoc-GFP distribution (left) and Mitotracker distribution (right) on two living HeLa cells during UV-induced apoptosis. Elapsed time following the UV treatment is shown in each panel. Scale bar, 20 µm. (G-I) Magnified Mitotracker images of the upper cell shown in (C,D,F). These panels show the detailed structure of mitochondria before (G), during (H) and after (I) cytochrome c release. Scale bar: 4 µm.

View larger version (26K): [in a new window]   Fig. 4. (A) Dynamic change of cytoc-GFP distribution (unbroken line) and average mitochondrial diameter (broken line) in a single living cell during UV-induced apoptosis. Data are based on analysis of images obtained from the upper cell shown in Fig. 2. Fmito/Fcyto represents the fluorescence ratio of cytoc-GFP between mitochondria and cytoplasm. (B) Time-dependent changes of cytoc-GFP distribution and mitochondrial diameter as measured from a single mitochondrion (marked by arrowhead in Fig. 2D,E).

View larger version (54K): [in a new window]   Fig. 5. Comparison of cytochrome c distribution (left) and morphology of mitochondria (right) in cells stained with anti-cytochrome c and Mitotracker Red. (A) Before the UV-treated cell entered apoptosis, cytochrome c was distributed mainly in mitochondria, which had the normal filamentous shape. (B) In a small percentage of cells undergoing UV-induced apoptosis, cytochrome c was released into the cytosol while mitochondria still remained in filamentous shape. (C) In some UV-induced apoptotic cells, cytochrome c was released into the cytosol and the mitochondria became swollen. (D,E) Results similar to those in B were observed in apoptotic cells induced by TNF (D) or actinomycin D (E) treatments. Scale bar: 20 µm.

View larger version (70K): [in a new window]   Fig. 6. Simultaneous confocal measurements of cytoc-GFP distribution (left) and Mitotracker distribution (right) in a living cell treated with CCCP (10 µM). Elapsed time following the CCCP treatment was shown in each panel. Scale bar: 20 µm.

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