Two kinds of phosphorylation of Drp1 are known to be involved in Mito fission (phosphorylation at ser16 and de-phosphorylation at ser637). HOWEVER, it depends  to the kind of stress do you want to perform because phosphgorylation at ser616 is not systematically observed.

I use temperatural stress on mice. It is ser637 that I am looking at and I observe significant changes.

Ok. In my opinion, no reports describe phosphorylation at ser616 during heat shock. So ser637 is the good choice!

Hi Pawel,

generally speaking I would recommend fluorescence microscopy to analyse the mitochondrial morphology in your samples. TEM needs usually much more effort (although it is the method of choice if you would like to study whether ultrastructural features (e. g., membrane morphology) are affected). If you have the resources I would recommend that you also check fusion factors (e. g., Mfn1/2, OPA1) so you get a more complete picture on the regulation of mitochondrial dynamics.

Hope this helps.

Regards, Christian 

Thank you Christian,

I did consider Mfn1/2, OPA1. I guess that analysis is inevitable.

Thank you all for your responses

Hi Pawel, 

I would recommend the fluorescent microscopy as Christian suggested. Confocal microscope works better with tissue slices. For the dynamics proteins, I would check both phospho- DRP1. The S637 is more characterized, but the S616 responds to oxidative stress, which did not capture enough scientific research for some reason. Cell Signaling has 2 good antibodies for both proteins. Importantly, OPA1 responds to heat shock at two levels, processing of the long isoforms to the short ones (which is easy to analyze by Western blot) and oligomerozation which needs a special western blot procedure. Here is the BBA paper about it http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686154/

Still, microscopy will be needed to proof whether the changes in dynamics proteins result into alteration in mitochondrial morphology.

Good luck :)

Thank you Haitham,

OPA1 Abs are on their way :)

All the best

Hy,

probably I'm redundant with my colleague, but if it is possible you would have to perform fluorescence analysis ( in vivo will be perfect.. obviously it depends from the tissue and from the microscope).  Moreover,  please,you should take care that phoshoDrp1 or OPA1 analysis are not equal assay. In my opinion, if you perform a stress event you will induce the activation of OMA1, and consequently the analysis of OPA1 is more conclusive compare to DRP1 phoshorilation.  I'm not an expert of DRP1 phosphorilation and I don't know if it was demonstrated the correlation between phoshorilation and stress, for sure it was demonstrated the relationship between stress and OPA1 processing.

Regards,

Francesco 

Thank you Francesco,

I am dealing with mice models lacking any fluorescent tags, while I have good collaboration with TEM experts. Moreover adipose tissue is not easiest to perform fluorescent analysis on. I will include OPA1 for sure now.

Regards

Hi Pawel,

Glad that you found the answers helpful. As you are planning for TEM, here is a paper that describes a change to mitochondrial cristae organization as a response to metabolic stress. The process is regulated by OPA1 oligomers which also responds to heat shock. You might find your data relevant.

Good luck

http://www.ncbi.nlm.nih.gov/pubmed/25298396

Awsome! Thank you