Hi all,
I started studying autophagy and found it is very complicated mechanism.
I got some concepts about autophagy as followed:
-when autophagic process is activated, LC3 I is converted to LC3 II so we can regard LC3 II level or LC3 II/LC3 I ratio as activation marker of autophagy.
-rapamycin is an mTOR inhibitor, so induces autophagy
-bafilomycin inhibits maturation of autophagosome, so inhibits autophagy
So I thought that if I induce starvation or treat rapamycin to cells, LC3 II level would go up, while bafilomycin would reduce LC3 II conversion. However, I found that both rapa and bafilomycin treatment increases LC3 II level.
I cannot understand these phenomenona at all and I think I haven`t got the right picture of autophagic mechanism yet. Please help me with this problem.
Please be aware that LC3-II levels are tissue- and cell-context-dependent. Based on the cell type you study and the conditions of growth it might be possible not to detect any changes in the LC3 II to LC3 I ratio even after you have added autophagy-stimulating or -inhibiting drugs. In order to properly address your question it is necessary to know the cell type you are working with and the cultivation conditions.
Side-note: I agree that getting into the autophagy field is certainly challenging. But when you get familiar with these pathways it will become a very rewarding area for research. Don't give up!
Best regards, Christian
There are many references available including the ''autophagy guideline'' but in short:
Rapamycin will induced LC3-I to II conjugation, but the LC3-II is also a traget of autophagic degradation and normally will disappear if you do not block autophago-lysosomal degradation (for example with Bafilomycin). While rapamycin induces formation and turnover of LC3-II, Bafilomycin will prevent turnover and lead to accumulation.
May be this explains.
Bafilomycin is late phase autophagy inhibitor, thus pretreatment with bafilomycin will cause accumulation of LC-II. This observation can be considered as reserve of autophagy and thus it will prevent the cell death to certain extent.
Upon the fact that bafilomycin inhibits maturation of autophagosome, so inhibits autophagy, then you should see the real effect of it by applying bafilomycin on autophagy-induced cells. At that time, if you see accumulation of LC-II again instead of LC-II to LC-I conversion as reverse manner of rapa, then it should be tissue- or cell-specific phenomenon.
Sercan
@ sercan, if u induce autophagy and block autophagosome acidification by bafilomycin it will again lead to LC3-ll accumulation. Here u shud consider LC3-ll as an autophagy substrate indicating blockade of autophagy
To clarify the picture I would suggest to test in paralel the levels of p62 that go down when autophagy is at play.
FYI: http://www.ncbi.nlm.nih.gov/pubmed/24710484
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