Lecture 5 Flashcards
(38 cards)
What is the interplay between EGF signalling and endocytosis?
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.
What is likely to occur when ESCRT proteins are mutated and why?
Give rise to cancer as they are involved in the degradation of activated Rs into lysosomes
Design an experiment to see where signalling occurs in the cell.
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.
When looking at cells treated with EGF and insulin, what results were observed?
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
What happens when there is too much TGFB signalling?
Cells undergo etm which is a hallmark of cancer
What happens when there is dysregulated signalling?
carcinogenesis and fibrosis
What is the role of TGFB signalling?
Cell growth and differentiation
Why is the endosomal environment conducive to TGFB signalling?
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.
Why was the activation of transcription by TGFB signalling thought to be dynamin dependent? What does this mean?
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
Why were cells treated with Epsin15 DIII?
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.
What was the control when cells were treated with Epsin15 DIII?
they were treated with EPsin15 DIII delta2 = doesn’t interfere with CME
Other than the use of Epsin15 DIII, how else was it confirmed that TGFB signalling was CME dependent rather than caveoli dependent?
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
What is the outcome when TGFBRs enter via CCP?
Signal transduction pathway is mediated = they enter early endosomes and can phosphorylate smad 2 that is recruited by SARA to the endosomal membrane
What is the outcome when TGFBRs enter through caveoli mediated endocytosis?
Degradative pathway is mediated = R will be diverted rom endosome and will reside in caveoli positive vesicles and be directed to degradation
Why is route of receptor entry important for modulating the signalling pathway?
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
How does TGFB signalling from caveoli occur? And what does this mean?
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
What happens to snail mRNA activity when you inhibit tyrosine kinase activity of TGFBR?
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
What happens when you KO Shc?
Huge increase in snail activity in absence of TGFB, even bigger in presence. No snail activity when tyrosine kinase activity of TGFBR inhibited
What is observed through microscopy of epithelial layer when TGFB is added (and for tyrosine K inhibition and Shc KD)?
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.
When using GFP of certain cell components, what is observed during TGFB signalling on an epithelial layer?
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
What effect does Shc KO have on cell invasiveness?
Huge increase in invasiveness = Shc is an important regulator on whether cells adopt EMT
What are the roles of Epsin?
It is an adaptor of EGFR internalisation. Recognises U cargo.
related to the dimerisation of E cad - if overexpressed =enhanced migration
What is the relationship between Epsin3 and breast cancer?
It is overexpressed in 40% of cases therefore is used as a hallmark for disease
How is the relationship between Epsin3 and TGFB signalling tested?
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.
