In a recent post, I was musing about why the SmartFlares weren’t in the swollen chloroquine-induced vacuoles. Out of interest more than anything, I did a quick experiment to see what would happen if I pre-labelled the endocytic pathway with one coloured dextran, then added in a second with the chloroquine.
Data are date stamped [2015-06-19].
I have to admit, I was expecting 100% yellow vesicles. As it happens, the thresholded Manders’ coefficients are 0.52 and 0.36 for M1 and M2 respectively (where M1 is the amount of Red overlap with Green and M2 is the opposite). I used JACoP to make these measurements using default levels of thresholding.
Here, the chloroquine is added with the second dextran. It was maybe worth chasing the first dextran into only lysosomes (by incubating for 60-180 minutes to clear the earlier compartments) but that’s hindsight for you.
The most obvious thing about the chloroquine-treated cells is the lack of green dextran, supporting the idea of endocytic inhibition. It’s also really clear (and actually quite pretty) that the swollen vesicles are the pre-formed lysosomes. A magnified view is shown below.
Impact on SmartFlares
So with that (n=1) experiment done. What does that mean for the interpretation of the SmartFlare data?
Previously, I had suggested that there was a good chance that endocytosis is inhibited in the presence of chloroquine. In support of this idea, there is less SmartFlare in the cells in the presence of chloroquine. Furthermore, these data provide no evidence for a chloroquine-mediated endocytic rupturing. If that were the case, I would expect to see both the red and green dextran in the cytosol (now that we’ve pre-labelled compartments in red) and we don’t.
Unfortunately, this ultimately means that the SmartFlares are still in endocytic compartments (and therefore not subject to the DMOG-induced changes in VEGF reported previously).