This is a cell. It's crawling!
It's a fish keratocyte, and scientists study cells like this to understand
basic principles of how cells move;
and, one of the techniques that scientists use to study cell movement,
and lots of other cell behaviors,
is Fluorescence Recovery After Photobleaching or FRAP.
FRAP is a technique that uses fluorescence microscopy to
determine the mobility of proteins inside a cell.
So, scientists wanted to know how cells like this crawl.
When they used electron microscopy on fixed cells,
they saw a dense network of cytoskeletal filaments called actin.
Actin is a protein that stacks one on top of the other to form filaments.
What scientists eventually figured out, mostly through biochemistry experiments,
is that as the actin filaments at the front of the cell get longer,
that pushes the membrane out, causing the cell to stretch forward.
Disassembling the actin filaments of the back helps the back of the cell move forward,
and there you have it a cell that crawls.
The scientists wondered if the data they had collected from in vitro experiments
matched with what was going on in real-life cells.
But it's hard to study the inside of a cell when it's crawling.
That's where FRAP came in.
In this 2017 paper,
scientists used FRAP to study the protein dynamics of actin in living crawling cells.
But to understand the results you have to understand how FRAP works.
Scientists use FRAP to measure how much and how fast
specific proteins move around inside the cell.
Now, to do that they have to be able to see the protein,
which they do by tagging the protein with a fluorescent protein, like GFP,
or with a fluorescent chemical tag.
Then they can just use a fluorescence microscope, like this one,
to see where the proteins localize inside the cell.
These green lines represent many,
many, many, individual actin proteins all stacked together into long actin filaments.
The problem is when scientists take a picture or even a movie of a live cell like this,
you can't tell whether the proteins are stuck in place
or if they're rapidly exchanging with other proteins in the cell.
That's because individual proteins are too small to see under a light microscope,
so we can't tell if individual proteins are moving or not from a movie like this.
However, we can mark a subset of proteins and see if that subset of proteins moves.
And the way we do that is with photobleaching.
See, fluorescent molecules like GFP
must first be excited with light before they emit their own fluorescence.
For example, the microscope shines blue light on
cells which makes the GFP in cells emit green light,
which the microscope's camera can photograph.
It turns out that too much excitation light can permanently alter the fluorescent protein
so that it no longer emits light.
That's called photobleaching.
So, in a FRAP experiment, scientists use a special
kind of fluorescence microscope called a confocal microscope,
which lets them expose a tiny region of the cell to high-powered laser light.
That bleaches the proteins in that region, but not elsewhere.
The proteins are still there, they're just not fluorescent anymore.
Then, the scientists can use lower-powered light to excite the fluorescent protein and
take pictures over time to see what happens to the proteins that were not bleached.
To visualize what's happening inside the cell during a FRAP experiment,
imagine that each person in this field is one fluorescently labeled protein.
Now, if the proteins are cytoplasmic,
then maybe they're just diffusing around in the cytoplasm, like this.
If a scientist suddenly bleaches this region of the cell, the proteins are still there,
but they don't fluoresce anymore, so that region of the cell looks dark.
But since all of the proteins are still diffusing around, the proteins that are
still fluorescent will eventually diffuse into the region that was bleached and make it bright again.
In a cell, that looks like this. After photobleaching, the fluorescence quickly recovers.
Now, if instead of diffusing around in the cytoplasm the proteins are stuck somehow,
they're immobile, then when a scientist bleaches a region of the cell,
the dark region won't get brighter because the proteins that are still fluorescent are immobile.
They can't diffuse around and fill in that bleached area.
In a cell, that looks like this. After photobleaching,
the fluorescence doesn't really recover.
Scientists can quantify how much and how fast the fluorescence recovers after photobleaching.
Proteins that move faster have steeper fluorescence recovery curves.
The steepness of this slope is how scientists calculate
the rate of diffusion for the protein of interest.
And if proteins are immobile, fluorescence levels won't recover all the way to pre-bleached levels.
How much the fluorescence recovers,
tells scientists what percentage of the proteins are mobile or immobile.
So, you can see that scientists can learn a lot about how proteins move around inside the cell
just from the fluorescence recovery curve.
Remember the fish keratocyte paper?
Scientists used FRAP to measure the mobility of actin proteins inside crawling cells.
While they expected to find highly mobile actin at the front of the cell,
where the filaments are actively growing to make the cell move forward, they also found
considerable mobility for the actin in the filaments farther back from the leading edge.
That was surprising because it suggests that this filamentous network is actively
being built up and broken down all the time while the cell crawls.
FRAP is cool!
It's a technique that, using light, lets scientists answer questions
about how proteins move around inside living cells.
What questions might you answer using FRAP?