Lecture 27- Mechanobiology III

Question 1

Explain how integrins are involved in mechanotransduction

Answer 1

Integrins sense the ECM stiffness and translate this into biochemical signals (specially the activation of focal adhesions kinases, Rho)

Question 2

What can regulate filamentous actin and YAP/TAZ?

Answer 2

Rho

Question 3

What does stiffened ECM promote in cells?

Answer 3

Promotes the epithelial to mesenchymal transition

Question 4

How can the level of epithelial to mesenchymal transition be visualised and what difference would be observed between stiff and soft ECM?

Answer 4

Green immunofluorescene indicating the level of Snail

Snail indicated the epithelial to mesenchymal transition

Soft ECM: low levels of fluorescence, low levels of Snail, low levels of transition

Stiffened ECM: high levels of fluorescence, high levels of Snail, high levels of transition

Question 5

Why is a lack of epithelial to mesenchymal transition under soft ECM conditions are pre-requisite for tumour progression?

Answer 5

The mesenchymal cells loose their ability to stretch to each other which increases the chance of metastasis

Question 6

In which ways can inhibitors work to perturb cell-ECM interactions and/or cell signalling?

Answer 6

1. Inhibitors aim to delay the formation of epithelial to mesenchymal transition to reduce cancer progression
2. Inhibitors can interfere with ECM stiffness and reduce the crosslinking of ECM
3. Inhibitors can prevent integrins from binding to ECM or to components which affect the ECM directly and degrade components
4. Inhibitors can block the mechanotransduction itself

Question 7

Give some example of ECM/adhesion signalling inhibitors

Answer 7

1. TGF-beta inhibitors
2. MMP inhibitors
3. Losartan
4. LOXL2 inhibitors
5. Integrin inhibitors
6. Rho inhibitors
7. HA inhibitors
8. FAK inhibitors

Question 8

How does the adhesive area vary with differences in ECM stiffness?

Answer 8

Low ECM stiffness: cells don’t spread so occupy a small adhesive area

High ECM stiffness: cells spread and occupy a large adhesive area

The geometry of the cell/size of adhesive area is a physical cue which steers the cells behaviour by dictating its signal transduction

Question 9

Outline the core cascade of the YAP/TAZ- Hippo pathway

Answer 9

1. Mst1/2 positively regulates Lats1/2 which regulates via phosphorylated YAP/TAZ
2. Mst1/2 can be regulated by a number of membrane and cytoplasmic regulators
3. YAP/TAZ is negatively regulated when phosphorylated
4. This is because the phosphorylation leads to increase degradation via the proteasome and the phosphorylation on serine generated a binding site for a 14-3-3 protein
5. 14-3-3 protein binds specifically to phosphorylated YAP/TAZ and prevents the transcription regulator entering the nucleus so its retained in the cytoplasm
6. When the pathway inactive, YAP/TAZ not phosphorylated so is active in the nucleus so it’s transcription regulator must enter the nucleus to interact with TFs

Question 10

What does excess YAP activation lead to?

Answer 10

Organ enlargement/over growing phenotypes

Question 11

What happen if the YAP gene is mutated?

Answer 11

Prevents phosphorylation which is important for degrading YAP

If degradation is prevented, the protein is over expressed which leads to the overgrowing phenotype

Question 12

What cell or tissue properties regulate the Hippo pathway?

Answer 12

1. Apicobasal polarity
2. Mechanotransduction
3. Cell-cell adhesion
4. Contact inhibition

Question 13

What cellular functions are regulated by the Hippo pathway?

Answer 13

1. Proliferation
2. Cell survival
3. Cell competition
4. Stem cell maintenance
5. Metastasis
6. Regeneration

Question 14

What regulates YAP nuclear localisation?

Answer 14

YAP/TAZ is mechanosensitive and regulated by ECM stiffness

YAP/TAZ is inhibited at low ECM stiffness and activated at high ECM stiffness

Question 15

Where is activated and inactivated YAP/TAZ and what causes this?

Answer 15

At high ECM stiffness, YAP/TAZ is activated and localised to the nucleus

At low ECM stiffness, YAP/TAZ is inactivated so will be degraded if found in the cytoplasm

Question 16

Explain cell size regulating YAP activation

Answer 16

Cells that could spread out over a large area allowed YAP/TAZ to be activated in the nucleus

Cells that could not spread out over a large area inactivated YAP/TAZ so was not localised to the nucleus

Question 17

Why were cells grown on micro pillar and what did this determine about YAP/TAZ activation?

Answer 17

To determine if YAP/TAZ activation is due to the amount of ECM contact or geometry

Concluded YAP/TAZ activation and nuclear localisation is due to the actually spreading of the cells and not the amount of surface contact

Question 18

What 3 things regulate YAP/TAZ nuclear localisation

Answer 18

1. ECM stiffness
2. Cell size
3. Actin cytoskeleton and actin contractility

Question 19

What is regulating the activation of YAP/TAZ?

Answer 19

Regulated by the actin-myosin contractility of the actin cytoskeleton

Question 20

How does YAP/TAZ regulate stem cell differentiation into oesteogenesis?

Answer 20

1. Under high ECM stiffness, YAP/TAZ is active and stem cells undergo osteogenesis
2. Under low ECM stiffness, YAP/TAZ is inactive and stem cell differentiation is reduced

Question 21

What is the role of YAP/TAZ in adipogenesis?

Answer 21

YAP/TAZ suppresses adipogenesis (despite the stiffness of the ECM)