QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 10 June 2008
doi: 10.1242/jcs.024703

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhou, L. Articles by Chang, D. C. Search for Related Content PubMed PubMed Citation Articles by Zhou, L. Articles by Chang, D. C. Social Bookmarking                         What's this?

Dynamics and structure of the Bax-Bak complex responsible for releasing mitochondrial proteins during apoptosis

Department of Biology, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

View larger version (47K): [in this window] [in a new window]   Fig. 1. Translocation of Bax from cytosol to mitochondria was correlated with Cyt c release during UV-induced apoptosis. (A) Western blots showing that Bax translocation was temporally correlated with Cyt c release, caspase-3 activation and PARP cleavage in HeLa cells during apoptosis. Cytosolic (C) and membrane (M) fractions of HeLa cells were harvested at different time points following UV irradiation. The bands marked * were the loading controls. (B) Quantitative analysis of the western blot results as shown in A. (C) Western blot analysis comparing the changes of the cytosolic fractions of GFP-Bax and endogenous Bax in HeLa cells following UV irradiation. (D) Quantitative analysis of the western blot results as shown in C.

View larger version (80K): [in this window] [in a new window]   Fig. 2. The distribution patterns of GFP-Bax and mitochondrial morphology in UV-induced apoptotic HeLa cells. (A) In stage I, GFP-Bax was diffusely distributed in the cytosol. (B) In stage II, GFP-Bax showed a filamentous distribution pattern that was colocalized with mitochondria. (C) In stage III, GFP-Bax formed small complexes at the MOM. (D) In stage IV, GFP-Bax formed large clusters in mitochondria. Scale bar: 10 µm. (E,F,G) Magnified views of the boxed regions in B, C and D, respectively. Scale bar: 2 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Time-dependent measurement of GFP-Bax distribution during UV-induced apoptosis in live HeLa cells. (A-H) Sample records from a time-series of confocal measurements of GFP-Bax distribution (left) and Mitotracker distribution (right) on two live HeLa cells. The cells beginning to form small complexes of GFP-Bax are indicated with arrowheads. Scale bar: 10 µm. (I) Quantitative analysis of the translocation of GFP-Bax and Mitotracker fluorescence intensity change of Cell 2 shown in A-H. (J) Analysed results of another imaging measurement showing that the release of Cyt c from mitochondria correlates with the reduction of Mitotracker fluorescence intensity.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Comparison of the distribution of Bax and Cyt c in the same apoptotic cells. (A-H) Distribution of GFP-Bax (A-D) and endogenous Cyt c (E-H) within the same HeLa cells undergoing UV-induced apoptosis. (I-P) Distribution of endogenous Bax (I-L) and endogenous Cyt c (M-P) within the same apoptotic HeLa cells. The distribution of endogenous Bax and Cyt c was visualized by immunostaining. The arrowheads indicate cells in which Bax forms small complexes. Scale bars: 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 5. The dynamic changes in the distribution of CFP-Bax and Smac-YFP during UV-induced apoptosis. (A-E) Sample records from a time-series of measurements of CFP-Bax distribution (left) and Smac-YFP distribution (right) in a single live HeLa cell. The cell beginning to form small complexes of CFP-Bax and release Smac-YFP from mitochondria to the cytosol is indicated with an arrowhead. Scale bar: 10 µm. (F) Quantitative analysis of the translocation of CFP-Bax and the release of Smac-YFP in a single HeLa cell as shown in A-E.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Distribution patterns of YFP-Bak in UV-induced apoptotic HeLa cells. (A-C) In stage I, YFP-Bak localized at the MOM. (E-G) In stage II, YFP-Bak formed small complexes at the MOM. (I-K) In stage III, YFP-Bak formed large clusters in mitochondria. Scale bar: 10 µm. (D, H and L) Magnified views of the boxed regions in C, G and K, respectively. Scale bar: 2 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 7. The dynamic distribution of Bax and Bak during UV-induced apoptosis. (A) A sample record of the distributions of Cherry-Bax (shown in green) and YFP-Bak (shown in red) in a live HeLa cell at t=112 minutes 54 seconds after UV treatment. The merged image is shown in the right. Scale bar: 10 µm. (B-E) Magnified views of the sample records from a time-series of measurements of Cherry-Bax and YFP-Bak. Scale bar: 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 8. FRET analysis of the interaction between Bax and Bak. (A,B) A sample record of CFP-Bax distribution (shown in red) and YFP-Bak distribution (shown in green) in an apoptotic HeLa cell. (C) Merged image of A and B. Arrows labelled 1, 2 and 3 indicate three different types of aggregates that were formed by (1) Bax alone, (2) Bak alone and (3) Bax and Bak. Scale bar: 10 µm. (D) The three types of aggregates marked by arrows 1, 2 and 3 in panel C were magnified and are shown in the upper panels. Their corresponding images obtained by FRET measurements are shown in the lower panels. Line-scan measurements were conducted. (E-G) Results of line-scan measurements conducted on the FRET images shown in the lower panels of Fig. 8D. (H) A sample record of FRET measurement using the photobleaching method. YFP-Bak was photobleached in the region marked by a box. Line-scan measurements of CFP intensity were conducted along the lines drawn across the aggregates outside (arrow a) and inside (arrow b) the photobleached region. (I,J) Results of line scan measurements conducted on the CFP-Bax aggregate outside (I) (marked by arrow a in panel H) or inside (J) (marked by arrow b in panel H) the photobleaching region before and after photobleaching.

View larger version (41K): [in this window] [in a new window]   Fig. 9. Quantitative measurements of the size of the GFP-Bax complex and determination of the number of molecules within a single Bax complex at the MOM. (A) Image records of GFP-Bax small complexes in an apoptotic HeLa cell (left panel) and fluorescent beads (right panel) obtained under identical imaging conditions. Scale bar: 2 µm. (B) A sample image analysis of a 0.2 µm diameter bead using the MetaMorph software. (C) A calibration curve for converting the diameter from pixel numbers to µm based on the measurements of beads. (D) A sample image analysis of a GFP-Bax small complex. (E) Results of measurements on the size of GFP-Bax small complexes (n=316, from six cells). (F) Image record of purified GFP droplets obtained under identical imaging conditions as in A. Scale bar: 2 µm. (G) A calibration curve for converting the integrated fluorescent intensity to number of GFP molecules based on the measurements of GFP droplets. (H) Results of measurements on the number of GFP-Bax molecules within a Bax small aggregate (n=150, from six cells).

View larger version (37K): [in this window] [in a new window]   Fig. 10. A schematic showing the dynamic relationship between Bax-Bak redistribution and the release of Cyt c and Smac from mitochondria during apoptosis. There are four distinct stages of Bax-Bak distribution in an apoptotic cell. (Stage I) Before an apoptotic stress signal is activated, Bax is diffusely distributed in the cytosol, whereas Bak is localized at the MOM. (Stage II) After the stress signal is activated, Bax starts to translocate to mitochondria and distributes evenly at the MOM, whereas Cyt c and Smac remain in the mitochondria. (Stage III) Within a few minutes, Bax and Bak begin to form small complexes at the MOM by lateral diffusion along the mitochondrial membrane. Cyt c and Smac can now be released from mitochondria through the Bax-Bak complex. (Stage IV) The Bax-Bak complexes expand to form large clusters by recruiting additional Bax and Bak molecules.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter