spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 24 July 2008
doi: 10.1242/jcs.028977

Journal of Cell Science 121, 2671-2684 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Low, C. P.
Right arrow Articles by Yang, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Low, C. P.
Right arrow Articles by Yang, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Caspase-dependent and -independent lipotoxic cell-death pathways in fission yeast

Choon Pei Low1, Guanghou Shui1, Li Phing Liew1, Sabrina Buttner2, Frank Madeo2, Ian W. Dawes3, Markus R. Wenk1 and Hongyuan Yang1,3,*

1 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Republic of Singapore
2 Institute of Molecular Biosciences, University of Graz, Universitätsplatz 2, 8010 Graz, Austria
3 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052 New South Wales, Australia


Figure 1
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 1. Apoptotic and non-apoptotic cell death in TAG-deficient S. pombe mutants upon entry into stationary phase. (A) Viability of wild-type and DKO cells at various growth phases in rich medium. (B) Viability of wild-type and DKO cells over time in minimal medium. Values shown are means with standard errors (s.e.m.) from independent experiments (n>=3). (C-G) Nuclear-envelope (Nup124p-GFP) and chromatin (DAPI) morphology of wild-type and DKO cells at various growth phases in rich medium. (I-L) Nuclear morphology of wild-type and DKO cells in minimal medium. Scale bar: 5 µm. (H) Enlarged views of DAPI-stained DKO cells at: (left) early stationary phase in rich medium; (right) day 3 of stationary phase in minimal medium; and (middle panel, showing condensed metaphasic chromosomes) metaphase-arrested nda3 KM311 cold-sensitive mutant. Note the distinct DNA morphology. Scale bar: 2.5 µm.

 

Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 2. Fatty acids, DAG and ceramide induce distinct forms of lipotoxic cell death in fission yeast. (A-C) Lipid profiles of wild-type and DKO cells at logarithmic phase (cell density of 1x107 cells/ml) and early stationary phase (6x107 cells/ml) in rich medium. A.U., arbitrary unit (defined in Materials and Methods). *P<0.05; **P<0.001. (D) Cell viability following oleic-acid treatment. (E) Cell viability following DAG and sphingolipid treatments. *P<0.05. DHS, dihydrosphingosine; PHS, phytosphingosine. All values shown are means with s.e.m. from independent experiments (n>=3). (F) Nuclear morphology following various lipid treatments. Scale bar: 5 µm.

 

Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Lipoapoptotic cell death in rich medium is characterized by delayed plasma-membrane permeabilization and by post-mitotic arrest. (A,B) PI (A) and Phloxin-B (B) staining of wild-type and DKO cells at various growth phases in rich medium. *P<0.0005; **P<0.005. (C,D) Aniline-blue staining of septum (C) and septation indices (D) of day-1 stationary-phase cultures in rich medium. Scale bar: 10 µm. Values shown are means with s.e.m. from independent experiments (n>=3).

 

Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 4. Kinetics of molecular events during the induction of cell death in conditioned rich medium, which recapitulates stationary-phase conditions in rich medium. (A) Nuclear morphology after 90 minutes in conditioned rich medium. Scale bar: 5 µm. (B) Time course of viability in conditioned rich medium. (C-E) Lipid profiles of wild-type and DKO cells after 60 minutes in conditioned rich medium. A.U., arbitrary unit (defined in Materials and Methods). Values shown are means with s.e.m. from independent experiments (n>=3). *P<0.05. (F) Percentages of post-mitotic and septated cells in conditioned rich medium over time. (G) Percentages of DKO cells that were viable or showed condensed chromatin or a fragmented nuclear envelope in conditioned rich medium over time. (B,F,G) Values shown are means from independent experiments (n>=3).

 

Figure 5
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 5. Active role of mitochondria in lipotoxic cell death of S. pombe. (A) Effects of mitochondrial inhibitors and other pharmacological agents on the nuclear morphology of DKO cells in conditioned rich medium (2 hours). Scale bar: 5 µm. (B) Viability of DKO cells in conditioned rich medium in the presence of various pharmacological agents (2 hours). Addition of DMSO or ethanol (used to dissolve drugs) into the control (conditioned rich medium only) did not produce any observable difference. (C) Effects of potassium cyanide (KCN) on log-phase DKO cells in rich medium and in treatments with DAG and ceramide. (D) Rh123 staining for mitochondrial membrane potentials at various growth phases in rich medium. (E) Oxygraphs showing the oxygen consumption of early stationary-phase wild-type and DKO cells in rich medium (from two independent experiments). Oxygen-consumption rates were determined from the slopes of the oxygraphs of signal (nmol oxygen/ml liquid phase, y-axis) versus time (x-axis). Red arrows mark the addition of KCN to ensure the drop of liquid-phase oxygen concentration was specific to mitochondrial respiration. (F) Oxygen-consumption rates of wild-type and DKO cells at various growth phases in rich medium. Values shown are means with s.e.m. from independent experiments (n>=3).

 

Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 6. ROS accumulation takes place downstream of mitochondrial activities during cell-death induction in rich medium and conditioned rich medium. (A) Scatter plots of ROS-PI dual stainings of cells at various growth phases in rich medium. (B) Numerical representation of results from A. Values shown are means with s.e.m. from independent experiments (n>=3). *P<0.05. (C) Upper three panels: scatter plots of control experiments for the specificity of stainings. Lower two panels: scatter plots of ROS-PI dual stainings of cells after a 1-hour incubation in conditioned rich medium. Potassium cyanide (KCN) totally inhibited ROS generation in conditioned rich medium, indicating the generation of ROS being downstream of mitochondrial activities.

 

Figure 7
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 7. Pca1 undergoes proteolytic cleavage that is dependent on cysteine 270. (A) Western blot of extracts of cells overexpressing C-terminally GFP-tagged pca1+. From the left: lane 1, vector control; lane 2, pca1+-GFP; lanes 3-5, three independent clones of pca1+-GFP with cysteine 270 specifically mutated to alanine. The same extracts were immunoblotted with serum raised against the N-terminus of Pca1 (B, left panel). In both cases, smaller bands corresponding to proteolytic products disappeared upon the mutation of the active cysteine 270. Untagged Pca1 (B, right panel) corresponded to the estimated full-length protein (~47 kDa).

 

Figure 8
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 8. Roles of putative cell-death mediators in lipotoxic cell death of S. pombe. (A,B) Viability of the indicated TKO cells in rich medium. (C,D) Viability of the indicated TKO cells in minimal medium. {Delta}pca1, {Delta}rad9, {Delta}pck1 and {Delta}bzz1 TKO cells exhibited attenuated cell death as compared with DKO cells only in minimal medium. Deletion of pck2+ or apg6+, however, had no effect in both media. Values shown are means with s.e.m. from independent experiments (n>=3). +/*P<0.05; ++/**P<0.005; +++/***P<0.0005.

 

Figure 9
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 9. Schematic diagram of the multiple lipotoxic cell-death pathways in fission yeast. FA, fatty acids; PL, phospholipids. Broken arrows denote the putative genetic pathway, whereas solid arrows denote biochemical pathways identified in this study.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008