Lecture 5

Question 1

What is the interplay between EGF signalling and endocytosis?

Answer 1

The activated EGF R is endocytosed to early endosomes to late endosomes to intraluminal vesicles to MBV for degradation. The cytoplasmic domain usually recruits signalling molecules but when in MBVs the kinase domain is no longer accessible.

Question 2

What is likely to occur when ESCRT proteins are mutated and why?

Answer 2

Give rise to cancer as they are involved in the degradation of activated Rs into lysosomes

Question 3

Design an experiment to see where signalling occurs in the cell.

Answer 3

Treat cells with EGF or insulin. Fractionate them then isolate them into plasma membrane fractions or endosomal fractions. Perform western blot to see which domains have been tyrosine phosphorylated.

Question 4

When looking at cells treated with EGF and insulin, what results were observed?

Answer 4

Insulin treated cells = not much tyrosine phosphorylation observed
EGF treated cells = some tyrosine phosphorylation observed at the receptor and cell surface substrates
Much more observed in endosomal fraction - likely to be the downstream signalling targets of EGFR

Question 5

What happens when there is too much TGFB signalling?

Answer 5

Cells undergo etm which is a hallmark of cancer

Question 6

What happens when there is dysregulated signalling?

Answer 6

carcinogenesis and fibrosis

Question 7

What is the role of TGFB signalling?

Answer 7

Cell growth and differentiation

Question 8

Why is the endosomal environment conducive to TGFB signalling?

Answer 8

TGFB activates smad2 so that it may be translocated into the nucleus and activate genes.
The endosome is rich in PI3P which recruits SARA via its FYVE domain. SARA recruits smad2 to the membrane so that it can be phosphorylated by TGFBR.

Question 9

Why was the activation of transcription by TGFB signalling thought to be dynamin dependent? What does this mean?

Answer 9

low luciferase activity was observed in dynamin mutant cells. Higher activity in control cells and even higher in dynamin overexpressed cells. This means that there is an interference with caveoli and CME

Question 10

Why were cells treated with Epsin15 DIII?

Answer 10

Epsin15 DIII is an inhibitor of clathrin mediated endocytosis. It was used to distinguish if TGFB signalling requires CME or if it occurred via caveoli. Luciferase activity was reduced meaning that TGFB signalling requires CME.

Question 11

What was the control when cells were treated with Epsin15 DIII?

Answer 11

they were treated with EPsin15 DIII delta2 = doesn’t interfere with CME

Question 12

Other than the use of Epsin15 DIII, how else was it confirmed that TGFB signalling was CME dependent rather than caveoli dependent?

Answer 12

Used nystatin which is a caveoli mediated uptake inhibitor - enhanced luciferase activity and similar levels of phosphorylated smad as control cells.
depleted potassium levels which inhibits CME = decreased luciferase activity and levels of phos smad

Question 13

What is the outcome when TGFBRs enter via CCP?

Answer 13

Signal transduction pathway is mediated = they enter early endosomes and can phosphorylate smad 2 that is recruited by SARA to the endosomal membrane

Question 14

What is the outcome when TGFBRs enter through caveoli mediated endocytosis?

Answer 14

Degradative pathway is mediated = R will be diverted rom endosome and will reside in caveoli positive vesicles and be directed to degradation

Question 15

Why is route of receptor entry important for modulating the signalling pathway?

Answer 15

Low levels of TGFB = enter through CME and undergo recycling
High levels of TGFB = excess enter through caveoli
The signal switch between the 2 pathways is ubiquitination

Question 16

How does TGFB signalling from caveoli occur? And what does this mean?

Answer 16

Activates the Erk Map kinase pathway - regulated by Shc protein found at the membrane that recruits either smad or MapK. This means that the rate at which Rs move through different pathways modulates signalling

Question 17

What happens to snail mRNA activity when you inhibit tyrosine kinase activity of TGFBR?

Answer 17

In control cells, when TGFB is added there is an increase in snail activity (marker for emt).
Inhibited tyrosine kinase cells = decrease in both cases

Question 18

What happens when you KO Shc?

Answer 18

Huge increase in snail activity in absence of TGFB, even bigger in presence. No snail activity when tyrosine kinase activity of TGFBR inhibited

Question 19

What is observed through microscopy of epithelial layer when TGFB is added (and for tyrosine K inhibition and Shc KD)?

Answer 19

control = epithelial junctions breakdown and elongated cell phenotype arises
inhibit TK activity = cell layer maintained
Shc KD = disruptive cell layer and migratory phenotypes. More pronounced in TGFB presence. Effects prevented when TK inhibited.

Question 20

When using GFP of certain cell components, what is observed during TGFB signalling on an epithelial layer?

Answer 20

In absence = E cad seen forming E junctions, very little ECM deposition
in presence = loss of epithelium and E cad signalling, deposition of fibronectin in ECM (fibrosis)
Inhibit TK activity = junctional organisation maintained
Shc KO = loss of E cad, deposition in ECM and disruption of actin cytoskeleton. rescued when TK inhibited

Question 21

What effect does Shc KO have on cell invasiveness?

Answer 21

Huge increase in invasiveness = Shc is an important regulator on whether cells adopt EMT

Question 22

What are the roles of Epsin?

Answer 22

It is an adaptor of EGFR internalisation. Recognises U cargo.
related to the dimerisation of E cad - if overexpressed =enhanced migration

Question 23

What is the relationship between Epsin3 and breast cancer?

Answer 23

It is overexpressed in 40% of cases therefore is used as a hallmark for disease

Question 24

How is the relationship between Epsin3 and TGFB signalling tested?

Answer 24

Looked at Ecad internalisation - performed acid wash to remove antibodies from cell surface to see what was going on inside.
in absence of TGFB = little Ecad inside
In presence = more seen inside
Epsin3 overexpression in absence = Ecad still seen on outside but not as organised
Epsin3 overexpression in presence = huge increase in E cad internalisation.

Question 25

What happens when you look at Epsin3 and Ecad co-localisation between 0 and 90 minutes and what does it mean?

Answer 25

At 0 minutes there is colocalisation seen at the cell surface
90 minutes = less colocalisation due to sorting within the cell
therefore Epsin3 is inducing E cad internalisation and driving emt

Question 26

What happens to the invasiveness of a cell when conducting Epsin experiments?

Answer 26

Look at invasiveness of cells on matrigel when adding TGFB = not much change
If overexpress Epsin3 = become very invasive and even more so when TGFB is added

Question 27

What is a xenograph experiment and what happens when Epsin3 is KD?

Answer 27

Is when a tumour is injected into an ME mouse and invasiveness is observed.
KD Epsin3 = reduced invasiveness of tumour

Question 28

What does high levels of Epsin 3 mean?

Answer 28

Marker for poor prognosis and metastasis due to breaking down of epithelium resulting in a migratory phenotype

Question 29

Why do TGFA and EGF effect the EGFR differently?

Answer 29

EGF binds more tightly to EGFR meaning it is directed to the degradative pathway (lysosomes)
TGFA unbinds faster, allowing EGFR to be recycled

Question 30

What happens when EGF binds to EGFR?

Answer 30

They are bound tightly and Rab7 is recruited and phosphorylated leading to the ubiquitination of EGFR = transient signalling

Question 31

What happens when TGFA binds EGFR?

Answer 31

TGFA dissociates from R quickly, allowing RCP (Rab11 effector) to bind which promotes R recycling = prolonged signalling
interference of RCP effects signalling

Question 32

What happens to ERK activation when KD RCP?

Answer 32

in control = transient EGF signalling and prolonged TGFA

RCP KD = still see transient EGF signalling, but lose the prolonged signalling of TGFA

Question 33

What effects does RCP KD have on cell migration and proliferation? What does this mean?

Answer 33

EGF = minimal effects
TGFA = huge reduction in both
This means that endocytic machinery adapt differently to different signalling conditions

Question 34

What does it mean when you say qualitative differences arise depending on R location?

Answer 34

As R travels from cell surface to early endosomes to late, at each stage they interact with different signalling molecules which each can activate different gene transcription depending on the location of R

Question 35

Using NGF and EGF as examples, why is the rate at which receptors move through the cell important?

Answer 35

Both undergo ligand induced endocytosis however EGF has transient signalling and NGF has prolonged signalling. EGF travels to late endosomes, will be ubiquitinated then travel to intraluminal vesicles of MBS via HRS ESCRT 1-3 complexes. MVBs will mature into lysosomes.

Question 36

What happens to EGF signalling when reduce endosome fusion?

Answer 36

Use siRNA to reduce endosomal fusion e.g. EEA1 and other proteins. EGFR stays in the early endosome for longer and neural outgrowth and differentiation is observed (accumulation of B tubulin). Also reduced proliferation. This is due to prolonged EGF signalling

Question 37

What are Rab5 established membrane microdomains?

Answer 37

Rab% can establish fusion or signalling domains e.g. RABGEFs can set up signalling

Question 38

Why is it difficult to show that endocytosis regulates signalling in vivo?

Answer 38

Rab5 and dynamin have many profound roles across the organism e.g. APPL1 endosomal protein mediates Akt substrate specificiies and cell survival in vertebral development