Snapshot versus film

Most studies of cell death take snapshots. You treat cells or animals, wait a set time, then grind up the tissue and measure what is there. It tells you how things stand at that moment.

The problem is that apoptosis is a process, and parts of it happen in minutes. A snapshot cannot easily tell you whether something was blocked, delayed or slowed down.

The Wisconsin team made a film instead.

The cells

They used a human cell line called ARPE-19. These are retinal pigment epithelial cells, from the layer of cells that supports the light-sensing cells at the back of the eye. They are "immortalised", meaning they keep dividing in a dish, which makes them a reliable workhorse for lab research.

The choice fits the lab. It sits in an ophthalmology department, and SS-31 has been reported to protect eye tissue in earlier studies. It also means these are human cells, not rodent cells, which is useful for a mechanism study.

The cells were grown in standard conditions at body temperature.

Painting the parts in colour

The clever part is how they made three different things visible at once in the same cell. Using a technique that pushes DNA into cells, they introduced genes for three fluorescent tags:

  • BAX joined to a red fluorescent protein
  • Cytochrome c joined to a green fluorescent protein
  • A blue fluorescent protein that goes to the mitochondria and marks their shape

So in each cell, red shows where BAX is, green shows where cytochrome c is, and blue shows the mitochondria.

In a healthy cell, red BAX is spread through the cell fluid, green cytochrome c is tucked inside the mitochondria, and blue mitochondria form a network.

When apoptosis starts, red BAX gathers into bright spots on the blue mitochondria, green cytochrome c spills out and spreads through the cell, and the blue network breaks into pieces.

Triggering death

To start apoptosis, they used a chemical called staurosporine at 1 micromolar. Staurosporine is a standard lab tool for triggering intrinsic apoptosis. It reliably pushes cells into the BAX-driven death pathway.

Before adding it, cells were incubated with SS-31 at a range of concentrations, from 0.01 to 10 micromolar, or with a vehicle containing the same small amount of solvent but no peptide. That vehicle control matters, because it rules out the solvent itself as the cause of any difference.

The camera

Imaging was done on a spinning disc confocal microscope, a type of microscope that can take sharp images of thin layers within a cell quickly and with relatively little damage from the light.

Here are the numbers:

  • Images were taken every 2 minutes.
  • Each time point captured 20 to 30 thin slices through the cell, so the team could rebuild a 3D picture.
  • Imaging started 14 to 17 hours after staurosporine was added, when most of these cells go through apoptosis.
  • Every image was taken with the same laser strength and exposure, so the brightness could be compared fairly between cells.

The 3D images were analysed with specialist software that can measure the size of individual mitochondria and track the build-up of BAX over time.

What the film looks like

The paper includes a sequence of still frames from one cell treated with SS-31 and staurosporine. It shows the classic pattern:

  1. Just before BAX recruitment, BAX is mostly spread out, cytochrome c is in the mitochondria and the network is intact.
  2. Within a couple of minutes of BAX starting to gather, cytochrome c has already moved out into the cell.
  3. Mitochondria stay intact through the early stages of BAX gathering.
  4. Shortly after BAX is fully recruited, the mitochondria break apart.

The authors note that this pattern was typical for both SS-31 treated and vehicle treated cells.

That single sentence is really the heart of the paper. We will unpack it over the next three parts.

Why this method is strong

This approach answers the question a snapshot cannot.

For each individual cell, the team can see exactly when BAX started moving, how fast it gathered, how long it took, how much gathered in total, when cytochrome c escaped and when the mitochondria broke up. Then they can compare those numbers between SS-31 and vehicle.

If SS-31 really blocks BAX, it should show up in at least one of those measures. If it does not, the film will show that too.

How they handled the statistics

For two groups, they used a standard t-test. For several groups, a one-way ANOVA. To test whether SS-31 changed the share of cells that started apoptosis during the imaging window, they used a chi-squared test. Significance was set at the usual 5% level.

Next, the first result: what SS-31 did to the mitochondria before any death signal was given.

This series discusses a peer-reviewed laboratory study in cultured cells. The results have not been shown in people. Nothing here is medical advice, and SS-31 is discussed for research purposes only.