The double-edged sword of Damocles

August 23, 2010 § Leave a comment

Apoptosis is everywhere: it formed the spaces between your fingers and toes as you developed in utero, it prevents the development of T cells that would attack the cells of your body, and it shapes the structure of your brain.  Too little apoptosis can cause cancer; over-zealous apoptosis causes much of the damage in a stroke or heart attack, and contributes to the failure of organ transplants.  Not surprisingly, apoptosis is extremely tightly controlled at many levels.  The key event in the decision to go ahead and die is the permeabilization of the mitochondrial outer membrane (a.k.a. MOMP).  Cells given an apoptotic signal may wait for quite a long time before MOMP happens, and can be rescued if the conditions around them change; but once MOMP begins, under normal circumstances, there’s no going back.  Proteins released from inside the mitochondrion unleash the activity of the so-called “executioner” caspases, setting off a vicious cycle that ends with most of the contents of the cell being chewed up.  In fact, MOMP itself is usually enough to kill a cell, even when the downstream caspases are inhibited.

Usually, but not quite always.  Recently, it’s been noticed that MOMP alone is not always a death sentence.  If the caspases are inhibited, a few cells survive.  Galit Lahav pointed me to an interesting paper that shows that the cells that survive do so because they have a few intact mitochondria left, which can divide and gradually repopulate the cell (Tait et al. 2010 Resistance to caspase-independent cell death requires persistence of intact mitochondria. Dev Cell. 18 802-13. PMID: 20493813)

To look at the phenomenon of post-MOMP survival, Tait et al. started by developing a new way to visualize mitochondria.  In the past, MOMP has been detected on a single-cell level by imaging cytochrome c release from mitochondria, using GFP-tagged cytochrome c.  Using this system, it looks as if MOMP is an all-or-none event (in fact, this same group reached that conclusion 10 years ago).  The new imaging method is based on different proteins, Smac and Omi, which — unlike cytochrome c — are degraded in the cytoplasm when they’re released from mitochondria.  This degradation reduces the background fluorescence from released protein, making it easier to detect intact mitochondria, which stay bright.  And imaging methods have just got better in the last 10 years.

Using this new system of visualizing the release of GFP-tagged Smac and Omi from mitochondria, Tait et al. can now see that in about 25% of cells MOMP is partial: most of the mitochondria are gone, but a few can still be seen as a punctate pattern of Smac-GFP or Omi-GFP staining. These intact mitochondria also have a membrane potential, and they can divide; they’re functional mitochondria that have somehow escaped the initiation of MOMP.  But they’re only a minority of the mitochondria in the cell; most of the other mitochondria in the same cell are gone.

Are these remaining mitochondria required for cells to survive caspase-independent cell death?  Certainly there seems to be a strong correlation between cells that show incomplete MOMP and cells that can grow out new colonies.  Tait et al. sorted cells expressing Smac-GFP into low- and high-fluorescence populations, after first triggering MOMP.  The high-fluorescence populations were enriched for cells that still had intact mitochondria; this population also had a higher number of cells that can recover and grow.

The key question is, why do these few lucky mitochondria survive? The major pathway that triggers MOMP involves the Bcl-2 family of proteins, which includes both anti-apoptotic (e.g. Bcl-2) and pro-apoptotic (e.g. Bax) members.  Tait et al wondered whether the mitochondria that don’t undergo MOMP have unusual levels of these proteins.  They do: GFP-Bax normally translocates to mitochondria as MOMP begins, but the mitochondria that fail to undergo MOMP also don’t accumulate GFP-Bax.  They also have higher than normal levels of GFP-Bcl-2.  In a nice experiment aimed at showing that this is not just coincidence, Tait et al. take HeLa cells that overexpress Bcl-2 and have mitochondria labeled with YFP, and fuse them with HeLa cells not overexpressing Bcl-2, which have mitochondria labeled with mCherry.  Then they induce MOMP.  The mitochondria labeled with YFP are the ones that survive, suggesting that Bcl-2 overexpression protected them from MOMP.  Just to nail this down, they used a drug to inhibit the action of anti-apoptotic Bcl-2s.  All the mitochondria in cells treated with this drug MOMPed (and none of these cells recovered); the inactive enantiomer of the drug had no such effect.

There’s no obvious reason why one mitochondrion should have more Bcl-2 than the next mitochondrion, and it seems quite possible that this is just a matter of chance.  As I’ve discussed before, the Sorger lab has shown that there’s significant cell-to-cell variability in key apoptotic regulators — including Bcl-2 and Bax — so given Tait et al.’s results, a cell that’s at the top of the range for Bcl-2 but at the bottom of the range for Bax might be expected to have a particularly good chance of having a few mitochondria survive MOMP.  These variable apoptotic responses could be clinically important.  Both apoptosis inducers and apoptosis inhibitors are being explored in the clinic for a number of applications.  Understanding the factors that tip one cell over the edge but spare a neighboring cell may be essential if we’re to use the double-edged sword of apoptosis wisely.

Tait SW, Parsons MJ, Llambi F, Bouchier-Hayes L, Connell S, Muñoz-Pinedo C, & Green DR (2010). Resistance to caspase-independent cell death requires persistence of intact mitochondria. Developmental cell, 18 (5), 802-13 PMID: 20493813

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