I want to study the effect of a homologously expressed protein on necrotic cell death pathways as well as on autophagy in yeast S cerevisiae. I need to know the best candidate genes and proteins that will help me quantify the extent of induction or inhibition of these specific pathways.
Thank you Johan!! LC3B I think is for autophagy. Do you have any idea about the Necrotic cell death marker as well??
Please look for alarmins like HMGB1which is released by cell nuclei during necrosis. This is an authentic marker in yeast also. Nhp 6Ap is the mammalian homolog of HMGB1 in yeast. You can also look for ruptured and damaged nuclear membrane using electron microscopy.
Hello Shakri,
if you would like to know more on detecting autophagy in yeast (as well as in other organisms) I recommend "Guidelines for the use and interpretation of assays for monitoring autophagy" published in Autophagy 8(4) 445-544 (2012). This comprehensive article is freely available (e. g., http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883/).
Regarding the determination of necrosis you could think about the following strategies:
1.) Determination of 'colony-forming units'
2.) Using a dye like FUN1 (viability assay)
3.) Propidium iodide staining
There is also a nice review on this topic freely available: "Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae" published in J Appl Microbiol. 114(2):287-98 (2013). You can find it here: http://onlinelibrary.wiley.com/doi/10.1111/jam.12024/abstract
Good luck with your experiments!
Best regards,
Christian
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