Mechanobiology

Question 1

What is a) Mechanobiology b) Mechanotransduction c) Mechanosensing?

Answer 1

a) The study of how physical forces and changes in cell or tissue mechanics contributes to development, physiology, and disease.

b) Conversion of a physical force to a biochemical response (aka mechanosignalling)

c) When a protein or a cellular structure responds to a physical cue to initiate mechanotransduction

Question 2

What are the 4 stages of mechanotransduction?

Answer 2

1. Mechanosensing: adhesion receptors
2. Signal transduction: mechanical signal transduced along a linked network, cytoskeleton is often the force conduit.
3. Signal integration at nucleus: accumulation of signals over time, chromatin rearrangement/ nuclear pore opening.
4. Cellular response: microseconds to minutes (cell shape, fate, motility, growth).

Question 3

What are 3 examples of mechanotransduction in the human body?

Answer 3

1. Blood pressure autoregulation: A Ca2+ dependent response counters increased blood pressure/ flow by constricting blood vessels to increase resistance and decrease blood flow.
2. Epithelial actin cytoskeleton orientates itself in direction of fluid flow.
3. Hair cell bundles: stereocilia coordinated movement in response to soundwaves causes tension on tip links opening MET channels.

Question 4

What is the structure of a lung in a chip?

Answer 4

Epithelial cell layer and endothelial layer with membrane in between them, a chamber above/below as well as a chamber either side.

Question 5

What is a physiological set up for chip in a lung and why is this the best set-up?

Answer 5

> Having air in upper chamber while liquid in bottom chamber (as epithelial cells in alveoli would contact air while endothelial cells would contact blood which effects their cytoskeleton).

> There is a faster increase in transepithelial resistance with this set-up which shows the monolayer greater resembles that of a in vivo lung.

Question 6

What is the purpose of the 2 side chambers in lung in a chip?

Answer 6

> 2 side chambers allows us to add and release a vacuum, add a vacuum to stretch the membrane due to lower pressure, release the vacuum to contract the membrane again so can regulate how stretched the membrane is.

> Vacuum allows us to mimic breathing due to regulating stretch of membrane

Question 7

How could we use lung in a chip to investigate the immune defence response in the lab?

Answer 7

Added E.coli in epithelial chamber, observed neurotrophils adhere where endo cells opposite to where E.coli are, move through membrane to epithelial side and engulf E.coli on the epithelial.

Question 8

What is stress defined by and what is its units?

Answer 8

Defined as forced applied over a certain area, so units is N/m2

Question 9

What are the 3 different types of stress?

Answer 9

1) Shear: Stress that acts parallel to area, E.g. fluid flow parallel to endothelial cells

2) Compression: Pushing force (N)

3) Tension: Pulling force (N) e.g. actin cytoskeleton on ECM

Question 10

What is Strain defined as and its formula?

Answer 10

Strain = change in length/initial length
- ε
- unitless

> Refers to the deformation or change in shape of a material or system due to an applied force or stress (indentation on a cell)

Question 11

What is stiffness defined as and what is its units?

Answer 11

Stiffness= Stress/ Strain, Pa

Question 12

Do different tissues have different stiffness?

Answer 12

Yes different tissue have different stiffness.

Question 13

What is an example of a) very fluid b) intermediate c) very stiff tissues?

Answer 13

a) Brain and breast

b) Fat

c) Bone

Question 14

What is the effect of ECM stiffness on stem cells and how is this shown?

Answer 14

The environmental ECM stiffness plays a key role in guiding stem cell differentiation. Stem cells placed on a range of ECM stiffness differentiate into different cell types (e.g. very stiff ECM causes differentiation into bone cells).

Question 15

Why is it important to understand the effect of ECM stiffness on stem cell differentiation?

Answer 15

We can use this knowledge to create more stem cell therapies.

Question 16

How can ECM stiffness be used as a diagnostic, use an example?

Answer 16

Some diseases alter stiffness of tissue, so we can calculate the stage of the disease based off the ECM stiffness. E.g. Fibrotic liver, the liver becomes more and more stiff.

Question 17

What is an advantage of ECM stiffness diagnosis?

Answer 17

To normally diagnose diseases like fibrotic liver, a biopsy was needed, but by measuring ECM stiffness using an ultrasound is less invasive.

Question 18

What are 3 methods used to measure tension?

Answer 18

Micropipette aspiration, Atomic force microscopy, Optical tweezers.

Question 19

What is the principle of anatomic force microscopy indentation and what does it measure?

Answer 19

> Method to measure indentation (stress) at small level (We know the area which force is applied to which we can work out stress from.)

> The more the lever is pushed down, the more strain is applied, and this is measured by the angle of the laser which reflects off the lever. Change in reflection angle can be measured to measure strain while knowing the force applied (know force applied and the area to work out stress)

Question 20

How does ECM stiffness help with self-diagnosis of cancer?

Answer 20

As tumour tissue is stiffer than surrounding tissue, we can feel the tumour despite it being within another tissue (as It is stiffer than the surrounding tissue).

Question 21

What are 4 reasons to why tumour tissue is harder than surrounding healthy tissue and what does this favour?

Answer 21

1. Increased cell density in tumours due to highly proliferating nature, secreting excess ECM, both of which increase stiffness
2. Fibroblasts (cancer activating ones) surround tumour, have contractile actomyosin which contributes to stiffening
3. Cytokines can trigger tumour cells to release more ECM which contributes more to stiffness.
4. ECM secreted is abnormally cross-linked making it more stiff

All of which increase ECM stiffness and therefore increase tension on nucleus allowing YAP to enter and favouring epithelial to mesenchymal transition (EMT).

Question 22

How does ECM stiffness mediate the epithelial to mesenchymal transition of migrating cancer cells?

Answer 22

Tumour cell in multicellular context, low ECM stiffness/ tension low on nucleus from actin cytoskeleton -> disruption of cell-cell contacts during EMT (epithelial to mesenchymal transition) -> cells migrate out of primary tumour -> enters blood stream (unicellular context) where ECM stiffness increases/ tension increases on nucleus via actin cytoskeleton.

Question 23

What are 3 examples of Mechanosensors?

Answer 23

1. Piezo channels
2. Integrins
3. Caveolae

Question 24

What are Piezo channels and how is it activated?

Answer 24

Mechanosensitive ion channels mostly permeable to Ca2+. Connected to actin-cytoskeleton which pulls open the channel when under tension.

Question 25

What are Caveolae and how do they work to cause a biochemical change from a mechanical signal?

Answer 25

Caveolae are small invaginations of the plasma membrane which act as a reservoir for extra membrane. So, when a cell is stretched by ECM tension or shear stress (pulling of actin cytoskeleton with counterforce from ECM), the caveolae flatten out so the membrane doesn’t tear. Cavin molecules dissociate from the plasma membrane and go into the nucleus to control transcription (mechanical signal leading to biochemical change).

Question 26

How do integrins interact with the ECM?

Answer 26

> Force applied by ECM (actin cytoskeleton pulling on ECM) causes clustering of integrins called focal adhesions.

> Focal adhesions (integrin clusters) act as contact points for the cell to the ECM -> Integrin -> Bind to Talin -> Viculin -> Filamentous actin (cytoskeleton) connected to nucleus.

Question 27

What is Talin and how does it interact with the ECM?

Answer 27

> Talin is a multi-modular molecule, depending on the force applied by the ECM is can unfold its helicle structure different amounts

> When the helical structure of Talin is unfolded by force application, Vinculin is presented and can bind to F-actin. This joins the actin-myosin cytoskeleton of the cell to the ECM (allows for actin-myosin to pull on ECM to create counter pressure).

Question 28

Why does F-actin alone not have contractile activity?

Answer 28

F-actin is a structural element, so alone does not have contractile activity.

Question 29

What is the acto-myosin cytoskeleton and its function?

Answer 29

Myosin motor proteins are found in between F-actin filaments which creates force by myosin pulling the F-actin filaments to slide across each other.

Question 30

How does the ECM exert force onto a cell in 5 steps?

Answer 30

1. Actin-myosin cytoskeleton generates pulling force
2. Vinculin is recruited by Talin (both recruiter proteins), binding F-actin to Integrins (Focal adhesion points)
3. Pulling force is exerted on focal adhesion points (clusters of Integrins) which intern pull on the ECM.
4. The force generated by the cell on the ECM is transmits an equal and opposite force back on the focal adhesions, in accordance with Newton’s third law of motion.
5. The magnitude and direction of this force can affect cell behaviour, such as adhesion, migration, and differentiation. (if If the pulling force by actin/myosin is greater than the pushing (back) force from ECM, this leads to movement of the cell if ECM is stiff as generates greater tension)

Question 31

What is the effect of the act-myosin force being greater than the counter force of the ECM?

Answer 31

The cell would move (as tension is higher than the force pushing back, occurs on stiff ECM).

Question 32

What is the effect of epithelial cells when ECM is stiffened?

Answer 32

Cells form a more flat structure and start to migrate out: Snail protein is upregulated when EC is stiffened which is a key regulator for epithelial-mesenchymal transition (shows ECM stiffness is a contributor to tumour progression).

Question 33

What are 5 different points in the pathway of cell-ECM interactions or adhesion signalling which can be targeted for therapy strategies and examples for each?

Answer 33

1) ECM production
>TGF-beta is a molecule which upregulates collagen and fibronectin to produce more ECM; inhibiting TGF therefore makes ECM less stiff.
>MMP inhibitors, MMP proteases are needed for maturation of ECM, block these will decrease stiffness and amount of ECM available.

2) Cross-linking of ECM
>LOXL2 inhibitors decreases cross-linking of ECM, decreasing ECM stiffness

3) Integrin
>Integrin inhibitors like antibodies block integrin from binding with ECM

4) Downstream signal transduction
>Rho inhibitors, blocking Rho dependent kinase stops activation of actin-myosin cytoskeleton.
>Focal adhesion kinase (FAK) inhibitors, inhibits FAK needed for downstream signal transduction.

5) Posttranscriptional modification of ECM
>hyaluronic acid (HA) inhibitors, stops Glycosylation post-transcriptional modification of ECM so it cannot bind to Integrins.

Question 34

Which protein plays a role in controlling organ size, what does excess of this protein lead to?

Answer 34

YAP, excess YAP leads to organ enlargement due to excess proliferation

Question 35

What are the 4 steps to the Hippo pathway?

Answer 35

1. Upstream signals, such as cell-cell contact and mechanical forces, regulates the Hippo pathway. This leads to the phosphorylation of MST1/2 (mammalian Ste20-like kinases) and LATS1/2 (large tumour suppressor kinases).
2. MST1/2 activates LATS1/2
3. Activated LATS1/2 phosphorylate the transcriptional co-activator YAP (Yes-associated protein) and its paralog TAZ (transcriptional co-activator with PDZ-binding motif)
4. YAP/TAZ contained within the cytoplasm via cytoplasmic retention (bound to 14-3-3 molecule), as well as being degraded (due to being ubiquitylated) so cannot enter the nucleus to trigger cell proliferation and anti-apoptosis pathways.

Question 36

What type of gene is YAP?

Answer 36

YAP plays a role in development, for controlling organ size, it is an oncogene

Question 37

What is the role of the Hippo pathway?

Answer 37

The Hippo pathway can lead to degradation or cytoplasmic retention of YAP/TAZ, which prevents their nuclear translocation and activation of genes involved in cell proliferation and tissue growth.

Question 38

What is the effect of dysregulated Hippo pathway and why would it be dysregulated?

Answer 38

> Dysregulation of the Hippo pathway means YAP/TAZ are not contained in the cytoplasm by cytoplasmic retention or degraded and translocate into the nucleus to activate expression of genes. This upregulates cell proliferation and anti-apoptotic pathways

> Dysregulation occurs when ECM stiffness is high increasing tension on nucleus so YAP can enter e.g. when proliferating tumour cells secrete ECM

Question 39

When is YAP found in the nucleus and an example of when this would occur in the body?

Answer 39

> When ECM stiffness is high (leading to high tension on nucleus) as well as the cell being able to occupy a large area, as the Hippo pathway is inhibited so YAP can enter the nucleus.

> E.g. a tumour cell excreting ECM increasing its tension.

Question 40

How does Rho regulate YAP levels in the nucleus, what is the effect of blocking Rho?

Answer 40

Rho is an activator of actin-myosin cytoskeleton, this exerts pull on the ECM which exerts counter force on the nucleus allowing YAP to enter (if stiffness is high). If Rho is blocked then actin-myosin cannot generate pull, so YAP is not found within the nucleus.

Question 41

How do you test if actin-myosin cytoskeleton contractility effects YAP levels in the nucleus?

Answer 41

Place cells on elastic pillars and lower actin-myosin contractility (using RHo-inhibitors) and measure YAP in nucleus.

Question 42

How to test the effect of ECM contact area on YAP in nucleus?

Answer 42

> Cultivate cell on micropillars with ECM on, they behave like they are spread on a wide area despite only being in contact with small amounts of ECM(YAP in nucleus)

> Shows it is not the contact area with ECM that is important, but the morphology of the cell:

1. • When YAP is in a small area, YAP is excluded from the nucleus
2. • When YAP is in a spread out, large cell, YAP localises in the nucleus

Question 43

What cells do stem cells differentiate into when placed on a) stiff ECM b) soft ECM c) stiff ECM while inhibiting Rho, what evidence does this show for the role of YAP in stem cell differentiation?

Answer 43

a) Osteoblasts cells (bone making cells)

b) Adipose cells

c) Adipose cells, showing YAP is involved in stem cell differentiation as inhibiting Rho means YAP cannot enter nucleus (reflects the fact that YAP can enter nucleus on stiff ECM making bone cells but not on soft ECM making adipose cells).

Question 44

What is the effect of all cell spreading, stiff ECM, increased contractile forces on a cell?

Answer 44

All of these increase concentration of YAP in the nucleus (due to inhibiting the Hippo-pathway), cells have high actin-myosin contractility, promoting cell proliferation and stem cell differentiation into osteoblast cells.

Question 45

What is the effect of all confined cell adhesion, soft ECM, decreased contractile forces on a cell?

Answer 45

All of these lead to decreased concentrations of YAP in the nucleus (due to working Hippo-pathway), cells have low actin-myosin contractility, leading to decreased cell growth and decreases anti-apoptotic pathways as well as promoting stem cell differentiation into adipocyte cells.

Question 46

How do test the effect of actin-myosin contractility on a cell?

Answer 46

Use elastic pillars under the cell to replicate lower actin-myosin contractility (causing less YAP in nucleus) while using rigid pillars to show high contractility (causing more YAP in nucleus). And use Rho inhibitors which cause YAP to not be in nucleus as inhibit actin-myosin cytoskeleton

Question 47

Why does a) high ECM stiffness b) low ECM stiffness effect the tension generated by the actin cytoskeleton?

Answer 47

a) High ECM stiffness means it resists deformation and generates a greater pushing force on the cell membrane, so more resistance is generated by the actin cytoskeleton meaning more tension acts on the nucleus.

b) Low ECM stiffness is more compliant, it can be deformed more easily, so there is less resistance against cell movement or forces generated by the cytoskeleton; meaning the actin cytoskeleton generates less tension against the nucleus (as the ECM doesn’t create a strong counter force for the ECM to act on).

Question 48

What causes ECM stiffness?

Answer 48

The stiffness of the ECM is primarily determined by its composition and structure, including the types and abundance of ECM proteins, such as collagen and elastin, and the presence of cross-linking molecules.

Question 49

how can mechanical forces promote tumour progression?

Answer 49

1. highly proliferative tumour cells generate lots of cells, meaning it increases in density
   tumours lead to more ECM secretion, so excess ECM contributes to stiffness
   >The ECM is abnormally cross-linked to further increase stiffness.
2. cells such as fibroblasts surround the tumour with contractile activity, contributing to stiffening.
   >immune cells secrete cytokines, resulting in more ECM being secreted so more tumour stiffness
3. Increased ECM stiffness leads to increased tension on nucleus allowing YAP to enter.
4. YAP in the nucleus allows for the continued increased proliferation (maybe SNAIL enters too)
5. The increasing ECM stiffness eventually causes the epithelial to mesenchymal transition due to disruption in cell-cell contacts such as tight and adherens junctions, causing them to rely more on integrin interactions and therefore migrate into the bloodstream.

Question 50

what is the composition of caveolae?

Answer 50

> Caveolin 1 and 2 proteins insert into the plasma membrane to induce curvature and invagination

> cavin 1 and cavin 2 are peripheral proteins which detach from the plasma membrane

> Caveolae1 connects to the stress fibres via filaminA

> Cavin complex enters the nucleus

> when the actin cytoskeleton is pulled, it pulls the caveolae membrane invagination and flattens it out

Question 51

What is the effect on ECM stiffness on tension?

Answer 51

The more stiff the ECM the more tension is generated as actin myosin cytoskeleton can pull harder against the ECM without it releasing tension (cell moves) (Soft ECM gives more slack)