Lecture 26- Mechanobiology II

Question 1

What is an alternative to culturing on petri dishes where the stiffness can be altered and tailored?

Answer 1

Hydrogels (mix of bisacrylamide and acylamide)

The ratio will alter the stiffness. The more bisacryalmide, the stiffer the dish will be

Question 2

What contributes to determining MSC tissue fate?

Answer 2

Partially determined by the stiffness of the surrounding environment

Question 3

How can you culture MSC tissues to have different fates and how can they be indentified?

Answer 3

Culture on hydrogels with a soft matrix will form brain tissue/neuronal cells etc

Culture on hydrogels with stiff matrix will form bone cells

The type of tissues that forms can be identified using markers e.g neuro, myo or osteo markers

Question 4

What can be concluded from studies where cells cultured at different matrix stiffness will form different tissues?

Answer 4

ECM stiffness regulates stem cell differentiation

Question 5

Give 2 examples of diseases associated with an increase stiffness

Answer 5

1. Liver fibrosis

2. Cancer tumours (e.g breast cancer)

Question 6

How can an increase in ECM stiffness diagnose liver fibrosis?

Answer 6

The liver increases in stiffness in fibrosis

Fibrotic livers have an increase production and deposition of ECM stiffness which can be viewed and measured using an MRI/elastogram

Question 7

How can knowledge of tissue stiffness assist intervention and therapeutic decisions in patients with liver disease?

Answer 7

Non-invasive elastogram procedures can help identify the stage of chronic liver disease

Allows some patients whose disease is less severe to avoid liver biopsies

Question 8

How can tissue stiffness help diagnose cancer?

Answer 8

Palpitation of breast tissue can allow a lump to be discovered

A lump suggests the lump tissue is stiffer than the surrounding tissue

This understanding can aid the diagnosis and knowledge about the progression of cancer

Question 9

How do mechanical forces promote tumour aggression?

Answer 9

1. Tumour tissue increases the amount of secreted ECM. This leads to an increase in ECM deposition And an increase ECM stiffness around the tumour
2. Crosslinking between collagen and upregulation of crosslinking enzymes increases ECM stiffness
3. Fibroblasts also migrate to the tumour which differentiate into myofibroblasts which can contract and contribute to ECM stiffness surrounding the tissue

Question 10

What 3 things does tumour stiffness effect?

Answer 10

1. Proliferation
2. Migration
3. Metastasis

Question 11

Mechanical control of tumour cell fate- what happens under high tension/ECM stiffness?

Answer 11

1. ECM stiffness increases the number of focal adhesion kinase activation and increases Rho
2. YAP/TAZ are then activated which moves into the nucleus and regulate transcription and leads to upregulation of collagen
3. This leads to further increase in secretion of collagen and deposition

Question 12

How does matrix crosslinking force tumour progression?

Answer 12

By enhancing integrin signalling

Question 13

Give 5 techniques to measure cellular mechanical forces

Answer 13

1. Atomic force microscopy
2. Micropipette aspiration
3. Optical tweezers
4. Magnetic tweezers
5. Uniaxial stretcher

Question 14

Explain the technique of atomic force microscopy

Answer 14

1. Allows a measure of indentation on top of the cell
2. Centilever with a probe which touches the membrane and produces an indentation
3. Indentation can be measured using laser deflection to calculate how much force was applied to produce the indentation
4. Plot graph of indentation vs force applied
5. Graph allows the stiffness of the cell to be calculated

Question 15

Give 3 examples of mechanosensors

Answer 15

1. Piezo ion channel
2. Integrins
3. Caveolae

Question 16

Explain how piezo ion channels are involved in mechanotransduction

Answer 16

The translation of a mechanical signal (increase in membrane tension) into a chemical signal (ion influx through the channel)

Question 17

What are the 2 proposed models for piezo ion channels?

Answer 17

Model 1: the channel can induce curvature of the membrane and the stretching of the membrane causes the ion channels to open

Model 2: force from lipids and actin filaments push the membranes apart to open the ion channels

Question 18

Explain how integrins behave as mechanosensors

Answer 18

1. The point where integrins interact with the ECM forms focal adhesions
2. Talin interacts with the actin-cytoskeleton which are connected to stress fibres
3. The stress fibres transduce a force from the PM to the nucleus
4. This leads to the opening of nuclear pores or the rearrangement of chromatin and changes transcription
5. Talin partially unfolds when force/stress is applied and allows the binding of Vinculin
6. This leads to the stabilisation of the focal adhesions which mature into fibrillar adhesions by replacing Talin and Vinculin with Tensin
7. These changes lead to downstream signalling/mechanotransduction cascades

Question 19

How do integrin proteins change when there is no force and then force?

Answer 19

No force: Talin is folded and Vinculin cannot bind

Force: Talin unfolds and Vinculin binds

Therefore Vinculin is important for regulating the stiffness of the cells and responding to ECM stiffness

Question 20

What are caveolae and how to they act as mechanosensors

Answer 20

1. Caveolae are invaginations of the PM and flatten upon stress/increase in membrane tension
2. This flattening leads to the release of the Cavin complex
3. Cavin detaches from the caveolae and elicits downstream signal mechanotransduction cascades and alter transcription in the nucleus