L6 - Final Remarks

Question 1

describe membrane of autophagosome?

Answer 1

double membrane

Question 2

where on the membrane of autophagosome is Atg8?

Answer 2

both surfaces

Question 3

where on the membrane of autophagosome is Atg12?

Answer 3

outside only

Question 4

where on the membrane of autophagosome is Atg5?

Answer 4

outside only

Question 5

does Atg12 and Atg5 stay on the autophagosome when it is completely formed?

Answer 5

no, leaves after it forms

Question 6

what is the supposed explanation for how the autophagosome is remodeled?

Answer 6

Atg12-Atg5-Atg6 works with Atg8 to remodel membranes

Question 7

what is the supposed explanation for how the autophagosome is recycled?

Answer 7

Atg4 deconjugates Atg8-PE on surface of autophagosome to recycle upon autophagosome maturation

Question 8

how does selective autophagy work?

Answer 8

• autophagy receptors (proteins) bind and select specific substrates
• target them to autophagosome for encapsulation
• thought to bind to Atg8 on inside of autophagosome membrane

Question 9

p62

Answer 9

autophagy receptor protein that has a UBA domain that binds ubiquitinated proteins and targets it to autophagy degradation
- binds to LC3 (Atg8 for humans) inside autophagosome

Question 10

what is the best marker protein for monitoring autophagy and why?

Answer 10

Atg8

• always present on autophagosomes
• Atg8 upregulated when autophagy is induced

Question 11

what is the main marker protein used in autophagy assays in humans?

Answer 11

:LC3

Question 12

how can GFP-tagged LC3 be used to track autophagy?

Answer 12

estimate average number of autophagosomes by counting number of GFP-LC3 under fluor microscopy images

Question 13

what are the downsides of using GFP-tagged LC3 to track autophagy?

Answer 13

1) overexpression of GFP-LC3 can lead to aggregation of LC3

2) increased number of autophagosomes can also be due to reduced autophagosome turnover

Question 14

describe LC3-conversion assay

Answer 14

use WB to monitor the number of LC3-I (unlipidated) to LC3-II (lipidated)

Question 15

what are the issues with the LC3-conversion assay?

Answer 15

crudely quantitative because:

1) expression of LC3 varies amongst different cell lines
2) anti-LC3 Ab more sensitive to LC3II
3) LC3-II can be degraded by autophagy
4) increase of LC3-II can be due to upregulation of autophagy (more autophagosome synthesis) or reduced autophagosome turnover

Question 16

what are the two assays developed to monitor autophagic flux in mammalian cells?

Answer 16

1) LC3-turnover assay

2) GFP-Atg8/LC3 cleavage assay

Question 17

describe LC3-turnover assay

Answer 17

WB based assay

• adding lysosomal inhibitors (stop acidification inside lysosome)
• prevents LC3-II sent to autophagosomes from getting degraded leading to accumulation
• difference in amount of LC3-II between samples with and without lysosomal inhibitors represents the level of autophagic flux
• compare this difference under different conditions
• more autophagy = larger difference between amount of LCII in samples with and without lysosomal inhibitors

Question 18

describe the GFP-Atg8/LC3 cleavage assay

Answer 18

WB based assay

• GFP protein resistant to lysosomal protease degradation
• autophagic flux measured by the amount of free GFP generated when GFP-LC3 is degraded by WB with anti-GFP Ab
• more GFP = more autophagic flux

Question 19

what are the functions of autophagy?

Answer 19

1) maintain AA pool during starvation
2) prevent accumulation of aggregates (neurodegeneration)
3) anti-aging (degrade damaged and old organelles)
4) tumor suppression
5) clear intracellular microbes