Lecture 25 - Mechanobiology III

Question 1

What are the 3 types of mechano-sensors?

Answer 1

• piezo channels
• integrins
• caveolae

Question 2

What are features of piezo channels?

Answer 2

• piezo is mechanically activated by membrane/cortical tension
• they are responsive to stretch - 2 models - bilayer & intact cells
• induces curvature - curvature is flattened out inducing gap
• actin seems to facilitate the force opening the channel

Question 3

How is a piezo gene mutation associated with disease?

Answer 3

There are a number of mutations that could lead to disease - e.g. red blood cells show abnormal ionic content is connected to the osmotic pressure within the cell, which is measured by membrane tension, and therefore piezo channels

Question 4

What are integrins?

Answer 4

Integrins are cell adhesion molecules, transmembranes receptors. They have alpha & beta subunits, which are used to form an active integrin complex. There are different modes that can interact with the extracellular matrix - 1 is an INSIDE OUT mechanism. The integrins are in an inactive version to start with. An activating factor will bind to the inside of the molecule which will cause a conformational change and change the affinity for ECM.

Question 5

How do integrins act as mechano-sensors?

Answer 5

The formation of focal adhesion is proportional to extracellular matrix stiffness. The stiffness will lead to the formation of focal adhesions, as well as focal adhesion maturation. The protein composition involved in focal adhesion changes which usually involves stabilization. The focal adhesion are critical structures that come in contact with the ECM. The clustering of integrins can be seen in contact with the ECM, and the focal adhesions join the ECM with the actin cytoskeleton (F-actin, Talin & Vinculin). Talin & Vinculin bind bind to Filamentus-actin (a key component in the cytoskeleton). F-actin structures are modified into stress fibres (mini-muscles) and are attached to nucleus. These can change the shape of the nucleus.

Question 6

What makes up the actin cytoskeleton?

Answer 6

(F-actin, Talin & Vinuculin)

Question 7

What does Talin & Vinuculin attach to?

Answer 7

F-actin (filamentus-actin)

Question 8

How is Talin a mechanosensor?

Answer 8

Focal adhesion respond to forces that are exerted on them. 1 way to measure the force and translate them to biochemical changes is via Talin. Talin has an interesting characteristic, as it is a multi-domain complex, and when force is exerted, the domains unfold - when it reaches a certain threshold or force level, the unfolding of Talin domains triggers a biochemical response inside the cell.

Question 9

What does the Talin domain bind to?

Answer 9

The domain itself doesn’t bind to anything (if folded), however when unfolded, the Talin molecule opens the binding site for another molecule - Vinculin (only binds when Talin is partially unfolded).

Question 10

What does Vinculin attachment to unfolded Talin lead to?

Answer 10

The binding of Vinculin leads to the stabilization of the contact between the focal adhesion and the F-actin cytoskeleton.

Force exertion leads to enforcement of connection between actin cytoskeleton and focal adhesion.

Example of how forces can be converted to biochemical changes

Question 11

What is integrin-dependent adhesion and mechano-transduction pathway?

Answer 11

Acto-myosin arecontracting structures in our cells and act as mini-muscles. Responsible for keeping tension within a cell. Without these, the cells will collapse.

Integrins are key molecules to interact with extracellular matrix and sense ECM stiffness

Question 12

What are examples of inhibitors that disrupt cell-ECM interactions and/or adhesion signalling?

Answer 12

• Rho inhibitors
• HA inhibitors
• FAK inhibitors
• Integrin inhibitors

Question 13

What is increased stiffness a driver for?

Answer 13

Tumour progression

Question 14

How can inhibition of ECM stiffness occur?

Answer 14

• Inhibition of ECM stiffness can occur by changing different parts of sensing cascade.
• One way is through integrin inhibitors - antibodies that bind to extracellular domain of integrins & interfere with the binding activity of integrins to ECM.

Question 15

What are caveolae?

Answer 15

Invaginations of the plasma membrane
- caveolae composition and their links to stress fibres and the actin cytoskeleton
- caveolae flatten upon shear stress/increase in membrane tension

Question 16

How is caveolae an example of a mechanical to biochemical change?

Answer 16

Proteins associated with the caveolae are disassociated when it is flattened out then travels to the nucleus. It can then induce transcription.

Question 17

What is the Hippo pathway?

Answer 17

It is related to mechano-transduction. Part of this pathway uses mechano-transduction, but not that old.

It regulates organ size

• excess YAP activation leads to organ enlargement
• advantage with eye - don’t need to cut it open to see if something is wrong.

Question 18

Describe the hippo pathway

Answer 18

• Membrane & Cytoplasmic regulators feed into Mst1/2 regulation
• Mst1/2 kinases phosphorylate and activate the LATS1/2
• LATS1/2 kinases phosphorylation YAP/TAZ (inactivating them)
• they become inactive by sequestering them in the cytoplasm and promoting their degradation
• this prevents YAP/TAZ from entering the nucleus, where they would normally activate transcription of genes involved in cell proliferation and survival.

Question 19

What does a mutation in the hippo pathway therefore lead to?

Answer 19

Mutation will mean that downregulation cannot occur and the organ increases in size.

Question 20

What cells or tissue properties regulate the Hippo pathway?

Answer 20

• apicobasal polarity
• mechano-transduction
• cell-cell adhesion
• contact inhibition

Question 21

What cellular functions are regulated by the Hippo pathway?

Answer 21

• proliferation
• cell survival
• cell competition
• stem cell maintenance
• metastasis
• regeneration

Question 22

How does YAP/TAZ have a role in mechano-transduction?

Answer 22

YAP nuclear localization is regulated by ECM stiffness and cell size.

Question 23

Explain an experiment to show YAP nuclear localization is regulated by ECM stiffness and cell size

Answer 23

Isolated cell and put on either soft/stiff matrix:
High stiffness - YAP in nucleus
Low stiffness - YAP in cytosol.
It responds to stiffness (mechanical cue) in terms of its subcellular localization.

YAP is a transcriptional regulator.

Not only stiffness, but cell size can also regulate subcellular localization. Cells can spread over different area sizes. Localization of YAP into the nucleus is dependent on cell size

A number of inhibitors were used to check whether acto-myosin contractility was important. Rho activation and G-actin is important in YAP localisation in the nucleus.

To check for acto-myosin contractility, they used inhibitor contractility (Y27632 & Blebbist).

Blebbist (inhibits myosin directly).

Inhibition means that YAP isn’t in the nucleus

Question 24

How does YAP play an important role in ECM stiffness directed stem cell differentiation?

Answer 24

YAP is important for stem cells to differentiate into osteoblasts on a stiff matrix

Question 25

How does the transcription regulator YAP play a major role in mechano-transduction?

Answer 25

Stiff ECM & cell spreading = active YAP in nucleus. Promotes proliferation and osteoblasts differentiation.

Small spreading area & soft ECM - YAP outside of nucleus. Can’t activate transcription, growth arrest and BLOCK OSTEOBLAST DIFFERENTIATION and promote fat differentiation.

Question 26

What role do focal adhesions have on nucleus?

Answer 26

Focal adhesions pull on nucleus during stiff conditions, meaning nucleus is oval, changing the permeability on nuclear pores, allowing YAP in.