Lecture 21 Autophagy 1 Flashcards by Rosie Rudin

Define macroautophagy

A mechanism to digest IC material

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Why do cells need degradation x5

homeostasis 
signalling
removing damaged components
recycling nutrients 
reprogramming cells (differentiation)

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Name 2 mechanisms of degradation

The ubiquitin/proteasome system (UPS)

* Autophagy

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Name 3 flavours of autophagy

Macroautophagy
Microautophagy
Chaperone-mediated autophagy

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Briefly explain macroautophagy

autophagosomes fuse to lysosome

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Briefly explain microautophagy

Lysosome invaginates to capture the cargo material

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Briefly explain chaperone-mediated autophagy

Specific proteins are unfolded by HSC70 and threaded 1 by 1 threaded via LAMP-2A for degradation by the lysosome

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Which pathways are/are not lysosomal

Proteasome - not

Macro/chaperone mediated/micro autophagy - is

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Which pathways do/do not degrade individual proteins

proteasome - individual
macroautophagy - bulk
chaperone-mediated/micro - individual

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Which pathway is a major turnover route for short-lived proteins

proteasome

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Which pathway can remove whole organelles

Macroautophagy

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Which pathway turns over specific, long-lived proteins

Chaperone-mediated autophagy/microautophagy

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Molecules released by … can support what

the macroautophagy pathway

metabolism

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Which pathway has a relatively low capacity

Chaperone/micro

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Name the 4 functions of macroautophagy

Nutrient recycling
Cellular remodelling
Removal of damaged components
Killing IC pathogens

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Nutrient recycling:
When is autophagy rapidly unregulated?
This causes…

Under nutrient starvation

Causes non-selective bulk degradation of the cytosol

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What occurs in cells lacking autophagy vs WT in starvation

Death vs the WT which get smaller due as they digest themselves

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When do autophagy-deficient mice die

during neonatal starvation

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Give an example where autophagy is needed for survival

Cancer cells in solid tumours

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Autophagy is the only mechanism for…

the degradation of organelles

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Autophagy is essential for forming which cell types?

Erythropoiesis (red blood cell differentiation)

Removal of sperm-derived mitochondria in C.Elegans fertilisation

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Describe the process of erythropoiesis

reticulocyte denucleated

mitochondrial clearance

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You only inherit mitochondria from…

mother

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Why are components removed by autophagy

Cellular components accumulate damage over time

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examples of when damaged components needed to be removed by autophagy

mechanical damage in muscles damaged mitochondria selectively removed

How can mitochondria become damaged

CCCP decoupled the mitochondria respiratory train i.e. depolarises the mitochondria

What happens to the lysosomal capacity as we age

decreases

What disease is autophagy related to

Neurodegenerative and ageing

What causes age-related degeneration

Reduced autophagy

What cells are most susceptible to damage

Long lived neurons | metabolic muscle cells

Name the hypothesis associated to autophagy and what it states

The dietary restriction hypothesis: | Starvation/exercise leads to increased autophagy which in turn leads to increased damage repair

What could starvation prevent according to the hypothesis

prevent ageing

Eat2 mutant worms vs WT

Calorific restricted worms | Live significantly longer vs WT

How do we know eat2 mutant worms live longer via an autophagy dependent pathway

Removing autophagy in these worms has no affect on dietary restriction

What happens to the effect of dietary restriction on life span, the higher up through evolution you go

The less the effect is

How does dietary restriction increase autophagy

If starving, ATP levels drop. AMPK/SIRT1 detect to ATP/AMP levels and activate autophagy and inhibit TORC1 which inhibits autophagy

What does TORC1 recongise

Torc1 recognises amino acids. If cell is deficient in amino acids, you upregulate autophagy.

What pathogens can escape into the cytoplasm

TB | Salmonella

How are pathogens usually killed

Encased in a phagolysosome Fuses to lysosome Digestion

Why is the cytoplasm favourable for pathogens

They use nutrients here to grow | Macrophages cannot detect them

How does autophagy play a role in killing IC pathogens

Cells recognise the bacteria in the cytosol and targets them for autophagy

Effect of inhibiting autophagy in myocardium infection

TB bacteria can escape into the cytoplasm and grow unchecked leading to infection

Effect of inhibiting autophagy in MRSA

They grow less well as autophagy promotes their growth

Recycling nutrients causes How manipulate?

1. to survive starvation 2. helps cancer to grow So could inhibit autophagy to prevent cancer yet cells accumulate damage over time so will get mutations if not removed

Damaged protein/organelle removal causes How manipulate?

- Ageing - Muscular dystrophy - Neurodegeneration - Cancer Increase autophagy in all these cases

Cellular remodelling causes

- Erythrocyte differentiation | - Removing sperm-derived mitochondrial

Intracellular pathogen removal causes How manipulate?

- Tuberculosis - MRSA - viruses Inhibiting autophagy removes MRSA as cant hide in autophagosomes but makes more susceptible to TB

When/what/who were autophagosomes first discovered by?

EM 1963 Christian De Duve

When/who/how shown autophagy occurred in yeast?

1992 Yoshimori Oshumi - Nitrogen-starved protease deficient S.cerevisiae - In cells slightly mutated, starving them leads to accumulation of vesicles inside the vacuole

How many autophagy genes were identified?

1993 Performed genetic screen to identify 15 autophagy genes

Identifying the Atg genes allowed?

* Disruption of autophagy to investigate its functions * A start on dissecting how the machinery works * Observation of autophagy in live cells

Describe the 4 steps in the formation of autophagosomes

1. Initiation 2. Expansion into cup shapes (phagophore) – formed from fats using lipid transport 3. Cup shape closes (autophagosome) 4. Fusion with lysosome (autolysosome) using Syntaxin 17

How is the organelle made compared to most organelles?

Only organelle in the cell to be made de novo – every other vesicle is generated by budding off from the organelles

When are autophagosomes upregulated?

Starvation

Why are autophagosomes easy to visualise with EM?

It has 2 membranes

Where are autophagosomes formed?

At the ER

How does selective autophagy occur?

Ubiquitination | Protein aggregates

How are things targeted to phagosomes

Ubiquitination

What aids in selective autophagy

Adaptor proteins

How do adaptor proteins work

* Adaptor proteins have a ubiquitin binding domain on one end and an Atg binding motif on the other end (LC3) * This binds and targets specific proteins/organelles e.g. mitochondria to the phagosome

Are adaptor proteins vital?

No, some proteins directly bind to the Atg8, independent of adaptors

Why do ubiquitin protein aggregates accumulate?

Removing autophagy means they accumulate ubiquitin protein aggregates