MitoTracker Red and LysoTracker Green can be used to assess mitophagy by monitoring the colocalization of the 2 stains which happens only if mitochondria are engulfed in autophagosomes. Colocalization can then be reported by Pearson coefficient (PC). However, PC is correlated with both the location and the intensity of the fluorescence signal. So the PC will increase only if the two colors are colocalized AND both their intensities are proportionally correlated. This is ideal for measuring colocalization of two stable stains, however this is not the case in mitophagy. Engulfing mitochondria into the autophagosome will decrease the MitoTR fluorescence due to depolarization of mitochondria and/or degradation of mitochondrial proteins bound to MitoTR. This leads to less PC value complicating the interpretation of the results.
I wonder if there is a better colocalization indicator that report location of the two signals irrespective to their intensities?
Hello
To quantify colocalization I think in your case is better Manders coefficient, in that case you can distinguish the amount of one channel that is overlapping the other channel. Moreover this index is independent of intensity but images must be thresholded to obtain a good result. I used a lot in my works to quantify overlapping between mitochondria and ER and also mitochondria and LC3. JaCOP is a good plugin for image J to obtain all these index.
Good luck
Thanks for the great advice. I tried Mander's results on Imaris and I see the need for careful thresholding. There is a away to calculate the threshold automatically but it gives exaggerated results sometimes. I guess it needs some optimization but I think it is the way to go.
I would suggest to use mt-mKeima assay, which is very sensitive, simple, fast and reproducible. It can be easily quantified by FACS.
Dear Haitham El-Sikhry,
I agree with David preferring Manders coefficient to Pearson, since Manders born specifically to the imaging co-localization while Pearson coefficient born for other goal and then it was applied at imaging.
However, I have some concerns about imaging and mitophagy since mitophagy is a very dynamic event and for my experience, imaging it's not the best technique to measure it.
I suggest to you to perform, in parallel, other biochemical experiments with the appropriate inhibitors of autophagy.
regards
Francesco
Totally agree. Even with the mt-mKeima approach, which seems the most elegant, verification with pharmacological inhibitors/activators will always be beneficial.
Dear Haitham,
You should also consider that all the mitotrackers are membrane potential dependent to various degrees. That is how they are selectively sequestered into the mitochondria. So most of the mitotracker red signal should be removed shortly after the mitophagosome fuses with the lysosome It might therefore be better to use a probe that is not membrane potential dependent, for example overexpression of a fluorescently tagget mitochondrial protein. This is a good approach:
http://www.ncbi.nlm.nih.gov/pubmed/24176932
Thanks Morten for your input. I agree Mitotrackers are not the best for this propose although they provide easy fast staining which indeed depends on mitochondrial membrane potential. However, Mitotracker Red and Orange are considered the most stable inside mitochondria as they bind to mitochondrial proteins strong enough to make them detectable after cell fixation. This is good for detection but may has functional influence during long staining. In contrast, TMRM and TMRE are most sensitive to mitochondrial polarization with no protein interaction. This makes them more suitable for monitoring mitochondria for longer periods but they have much less value in observing mitophagy.
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