Lecture 6 - Autophagy

Question 1

What is autophagy?

Answer 1

Auto-phagy self-eating, at the subcellular level

Question 2

Autophagy, or cellular self-digestion, is a cellular pathway involved in

Answer 2

protein and organelle degradation

Question 3

Autophagy relies on

Answer 3

Dynamic rearrangement of intracellular membranes to allow an organized breakdown and recycling of cytoplasmic portions

Question 4

Protein classification according to turnover

Answer 4

Intracellular proteins can be classified into: short-lived proteins (half-life, 10–20 min) and long-lived proteins

Question 5

most short-lived proteins are

Answer 5

degraded by the ubiquitin-proteasome system

Question 6

most long-lived proteins are

Answer 6

degraded in lysosomes via the autophagic pathway

Question 7

More than 99% of intracellular proteins are

Answer 7

long-lived

Question 8

Autophagy- 1 of 2 degradative systems

Answer 8

• the ubiquitin-proteasome system (short-lived proteins)
• the vacuolar degradative pathway/lysosomal system, (long-lived proteins)

Question 9

non-selective vacuolar degradation process is a

Answer 9

highly conserved pathway within eukaryotes

Question 10

Three types of autophagy are known:

Answer 10

(i) chaperone-mediated autophagy (CMA)
(ii) microautophagy
(iii) macroautophagy

Question 11

Question 12

Question 13

Macroautophagy = Autophagy

Question 14

CMA:

Answer 14

selective motif tagged protein translocation directly through the lysosomal membrane

Question 15

Microautophagy:

Answer 15

trapping and engulfing of cytosolic regions by lysosomes

Question 16

Macroautophagy:

Answer 16

formation and accumulation of double membrane intermediate vesicles

Question 17

primary mechanism for cytoplasm-to-lysosome delivery

Answer 17

macroautophagy

Question 18

Macroautophagy (from here onwards referred to as Autophagy)
Definition

Answer 18

or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation

Question 19

Three basic steps can describe the autophagic pathway:

Answer 19

(1). Formation of isolation membrane and preautophagosome
(2). Formation of Autophagosome
(3). Formation of Autophagolysosome

Question 20

How is autophagy induced

Answer 20

Nutrient depletion
ER stress
mTOR kinase senses nutrient conditioninduction via Regulatory complex
ROS stress
Hypoxia
Toxic compounds
Radiation
High temperature

Question 21

mTOR-upstream sensor

Answer 21

mTOR is a sensor for amino acids and ATP (ADP/AMP) and can integrate hormone stimuli via the class I PI3-kinase/PKB pathway

it directly or indirectly causes hyperphosphorylation of the autophagy protein Atg13.
This protein modulates Atg1

Question 22

Induction

Answer 22

Increased mTOR activity inhibits autophagy

Question 23

mTOR affects downstream

Answer 23

protein/protein affinities

Question 24

When mTOR is inhibited

Answer 24

autophagosome formation is favored and kinase activity of Atg1 increases

Question 25

when mTOR is stimulated by nutrients

Answer 25

Atg13 becomes hyperphosphorylated which reduces its affinity to- and association with Atg1, autophagy not being favored

Question 26

LC-3, a key protein and structural component

Answer 26

Atg8/PE or its mammalian homologue, microtubule associated protein 1-light chain 3 (MAP1-LC3) is required as a structural component of the autophagosome and is therefore recruited to the autophagosome membrane

Question 27

Where in the cell localizes mTOR?

Answer 27

association of mTOR with the outer mitochondrial membrane, where it is ideally situated to sense changes in the ATP/AMP ratio

it also localizes with LC3-positive membranes, suggesting an involvement in the autophagosome maturation step

Question 28

Question 29

Question 30

2 conjugation machineries of autophagy proteins (Atg) are involved in the vesicle formation
How does it work?

Answer 30

Macroautophagy: cargoes are sequestered within a unique doublemembrane cytosolic vesicle, an autophagosome.

Sequestration can be either nonspecific, involving the engulfment of bulk cytoplasm, or selective
targeting specific cargoes such as organelles or invasive microbes

origin of the membrane is unknown (ER-membrane, de-novo synthesis or mitochondrial membrane are current models)

Fusion with a lysosome provides hydrolases. Lysis of the autophagosome inner membrane and breakdown of the contents occurs in the autolysosome

and the resulting macromolecules are released back into the cytosol

Question 31

Levels of autophagy

Answer 31

Almost all tissues have a basal level of autophagy, but to varying degrees

Question 32

Not all tissues respond equally with starvation induced autophagy

Question 33

How is autophagy measured?

Answer 33

Electron microscopy, the gold standard
Fluorescence Microscopy
LC3, Beclin, other Atg proteins
Quantification of cells with numerous LC3 punctae
Quantification of autophagosomes per cell

Question 34

Already fused? Or not yet fused? What is the fused proportion?
Co-localization analysis between autophagosomal and lysosomal signal.
How is it measured?

Answer 34

Western Blotting for key proteins
LC3 I/II
Beclin-1
p62

Question 35

LC3-II is specifically targeted to the

Answer 35

Atg12-Atg5-Atg16 complex and remains associated with the limiting membrane, the autophagosomes and the autophagolysosomes

Question 36

LC3-II is found both on the

Answer 36

the lumenal and cytosolic surfaces of autophagosomes
The lumenal pool is degraded after fusion with lysosomes (loss of signal with increased degradation activity), while the LC3-II on the cytosolic side can be delipidated and recycled

Question 37

In higher eukaryotes, LC3 is the only known protein that

Answer 37

specifically associates with autophagosomes and autolysosomes

Question 38

LC3-II levels correlate with

Answer 38

autophagosome number
HOWEVER: cellular LC3-II levels correlate with the numbers of autophagosomes in the cells at a snapshot in time

Question 39

Question 40

Question 41

Question 42

Normal flux (A) vs block in degradation (B) and additional fusion with endosome prior to fusion (C)

Question 43

How is flux measured

Answer 43

Question 44

Autophagy is crucial
Autophagy knock-out mice die within the first day of life

Question 45

An impaired autophagic machinery is associated with many pathologies

Question 46

Autophagy can lead to cell death

Answer 46

with autophagy = type II cell death

Question 47

Cell death with Autophagy is a physiologically

Answer 47

controlled mechanism

Question 48

Autophagy can be utilized as an avenue for

Answer 48

cellular demise, especially during development (programmed cell death)

Question 49

Question 50

Thinking points
Why are cells different in their basal protein degradation activity?