What is the difference between Regular Autophagy and Defective/Dysfunctional Autophagy in Molecular Level? Most importantly what does it even mean at molecular level other than the meaning we can derive from the phrase itself, that autophagy is not effective. A lot of papers do not seem to address the issue specifically, so I am a little bit confused about it. And most importantly how do you confirm whether the autophagy induced by any agent in just a regular autophagy or a defective/dysfunctional autophagy? I guess the conversion/expression of LC3-II occurs in both cases.Then how do you distinguish defective autophagy and regular autophagy?
Thank You
Hi Ananda,
in my research I used lysosomes inhibitor chloroqiune - a compound that increases pH in lysosomes. As a result lysosomes are not able to digest cargo delivered by autophagosomes any more. Using this method you can see if an increase in LC3 lipidation (so called "LC3 II") is a result of increase in autophagosome biogenesis or retardation of their digestion after fusion with lysosomes.
You can also try to look for p62/SQSTM1 abundance. It's a molecular adaptor that recognize Ub on proteins and directs them to autophagosomes. So if you have an increase in p62/SQSTM1 amount on the top of LC3 II increase, it means that autophagosomes probably are not able to cope with their duties. You can also assess the abundance of ubiquitinated proteins.
Ananda,
You might as well induce autophagy in your system where it is dysfunctional (say, you have one of the autophagy genes mutated) and compare it with control (non-mutant or corrected one) and get the LC3II/I ratio at different time intervals.
Thank you Dr Stanislovas S Jankauskas for a helpful insight.Now, I am wondering if I do a densitometric analysis of LC3 II and compare the compound treated group with bafilomycin treated and bafilomycin alone,then what value(probably a range) indicates the dysfunctional autophagy?
Thank you Alex Lyakhovich I will be doing it next.
Thank you Shounak Baksi
The paper seemed very detailed and helpful.
Ananda Baral , if LC3 II level in "Drug + Bafilomycin A" group wound not be higher than in "Drug only" group, than you don't have an increase in autophagosome biogenesis. However, you should use Bafilomycin A concentration, that capable of LC3 II increase in "Bafilomycin A only" group.
Thank you Dr Stanislovas S Jankauskas I checked that and the LC3 II is higher in case of "Drug+Bafilomycin A" than in "Drug only" group.This probably suggest autophagosome biogenesis. However,even though there is autophagosome biogenesis could there still be the existence of dysfunctional autophagy. As in my experiment there is increase in p62(whose expression level corresponds exactly to that of LC3 II in a time dependent manner) and not much difference in LC3II between (Drug+Bafilomycin A-Drug to that of Bafilomycin A-No treatment).
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Pharmaceutical Botany
- Plant Extracts