Hi there,
For the last few months I have been trying to measure the rate of mitochondrial fission and fusion after treatment with specific stress inducers. I use a hela cell line expressing a GFP tagged with mitochondrial Pre-sequence. I can see the fragmentation process when I am doing live cell imaging after pretreatment but I don't know how I can quantify this effect. I have done the same with immuno-staining but how can I measure the rate of fission versus fusion?
Thank you for your time and help!!
Dear Roy,
the quantification of mitochondrial dynamics is quite challenging. It can be done by using photoactivatable GFP that is targeted to the mitochondrial matrix. Here is a link to the paper where this technique has been applied first:
http://jcb.rupress.org/content/164/4/493.full.pdf
Maybe this article can give you some ideas on how to achieve your goal.
Best regards, Christian
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476419/pdf/jove-65-3991.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364104/pdf/nihms-367722.pdf
Two references with good approaches
Dear Roy,
For estimating fission, you can try to follow translocation of the fission protein Drp1 from cytoplasm to mitochondria by Western-Blot or by immunofluorescence. However, that's not so easy!!
you can also measure lenght of tagged-mitochondria by immunofluorescence by the ImageJ software.
Daniel
A Big thanks to every one.
I have gone through the publications and they seem quite helpful. I should have mentioned that i also created some photo-switching mEOS constructs targeted to the mitochondria. But i didn't quite know how to quantify the phenomenon.
I have begun quantifying the speed of movement of mitochondrial particles using a macro written for FIJI. Side by side I'm also using the 3d Object counter function to count the number of fragments and measure their surface area. The results seem quite promising but my concern is that I don't know if the methods I've followed are convincing enough.
I love the DRP1 idea and I've used Structured-IM to resolve the DRP1 rings at the mitochondrial break points but again I'm not sure if it's quantifiable!
However since this point was picked up, Is there a way to measure the GTPase activity of DRP1? Since this GTP hydrolysis is the step need for the act of fission, could we possibly measure this?
Thanks all of you again for your time,
Regards,
Roy
Hi there,
To my knowledge, change in mitochondrial dynamics can be studied by measuring the mitochondrial area and aspect ratio (major axis/minor axis of mitochondria). Also, in absence of unaltered mitochondrial biogenesis, fission results in increased number of mitochondria which can also be measured and presented. However, I am not sure if you want to specifically measure the rate at which these processes are happening then you would like to do live imaging on your samples.
Regarding GTPase activity, it is also required for fusion process so your results may not be very conclusive. You can either look at the transcript levels of fusion/fission genes or of the proteins present in the inner/outer membrane that are known to regulate assembly or stability of fusion/fission proteins. That's a suggestion if it may work out for you.
Best,
Hi Mamta,
That part about the GTPase activity is good advice! I didn't think the fusion part through however I do have plans for the qRT pcrs ready.
As far as the other points go I agree with all of them. As i mentioned before i'v counted the number of mitochondrial fragments and then compared the surface area of the un-fragmented mitochondrial network to the fragmented ones using the 3D object counter from FIJI.
For live cell imaging, I took images every 15 seconds for 20 minutes and then tried to measure the distance covered by one fragment from T=0 to T=20min using the macro script. But as i said, I don't really know if this is valid.
I mean this might really tell me how fast they move not the rate of fission/fusion because i have a feeling that that my treatment is slowing down the motility of the mitochondria compared to control cells and i do not know if this is because of lack of fission/fusion events.
Thank you for your time to reach out!
Best wishes,
Roy
I believe so too. We wrote the macro to follow the mitochondria in termsofspatial motility but I guess i can tweak it to count fragments per frame. Or else do it manually by analysis of individual time point frames.
Thanks again Mamta!
Best wishes,
Roy
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line