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Mechanobiology Flashcards

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1
Q

what is mechanobiology?

A

Mechanobiology: the study of how physical forces and changes in cell/tissue mechanics contribute to development, physiology and disease

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2
Q

what is mechanotransduction?

A

Mechanotransduction: the conversion of a physical force to a biochemical response (aka mechanosignalling)
- Stiffness of ECM can alter cell behaviour/proliferation

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3
Q

what is mechanosensing?

A

Mechanosensing: when a protein or cellular structure responds to a physical cue to initiate mechanotransduction
- How does a cell know if it is on a stiff or soft ECM?

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4
Q

what are the key concepts of mechanotransduction, in order?

A
  1. mechanosensing
    - cells test their environment via adhesion receptors (integrins) and membrane proteins probing the ECM
  2. signal transduction - mechanical signal transduced along cytoskeleton network
  3. signal integration at nucleus - accumulation of signals overtime to integrate info
    - leads to chromatin rearrangement, nuclear pore opening
  4. cellular response - microseconds to minutes
    - alters cell shape, fate, motility, growth
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5
Q

how is mechanotransduction involved in blood pressure?

A

Blood pressure autoregulation and coronary artery disease – myogenic tone
- body must keep blood pressure constant: mechano-response to pressure
- as pressure increases to 60mmHg in vessel, there is a calcium-dependent physiological response, where the diameter of the arteriole contracts
- arteriole lumen becomes smaller to limit blood flow and thus decrease pressure

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6
Q

what happens to cells upon fluid flow?

A

they change their cytoskeleton:
- Flow of fluid (strain) over a cell is a force exerted parallel to the cell surface
- endothelial cells under fluid flow strain results in actin stress fibres aligning in a parallel fashion to the fluid flow
- cell recognises direction of fluid flow and alters direction of cytoskeleton accordingly

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7
Q

how is mechanotransduction involved in the auditory system?

A

Stereocilia move when sound reaches the ear due to movement of fluid:
- Fluid exerts force on stereocilia
- Stereocilia bend, causing the opening of ion channels

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8
Q

what is a lung on a chip?

A
  • device which has small channels to emulate fluid flow through vessels
  • emulates contraction and extension of alveoli at the air-fluid interface
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9
Q

what is the structure of the cells of an alveoli?

A

Alveoli are composed of epithelial cells which face the air, and endothelial cells which face the capillary

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10
Q

how does the lung on a chip emulate alveoli function in breathing?

A
  • The system generates epithelial cells in close proximity to endothelial cells via a membrane in middle chamber made of PDMF with small pores in it and is flexible
  • Top channel faces epithelial cells, bottom channel faces endothelial cells
  • Porous membrane in the middle
  • 2 side chambers are key in emulating mechanical activity
  • Side chambers allow vacuum to be added and released
  • Can pump out the air to decrease pressure and allow membrane to stretch
  • Can release the vacuum to increase pressure and allow membrane to contract
  • Can define how much membrane stretches by how much of a vacuum there is
  • can easily mimic breathing activity
  • enables mimicking of lung function in infection/disease
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11
Q

how was the accuracy of the lung on a chip regarding physiological function tested?

A
  • immunofluorescence of epithelium (occludin) and endothelium (VE-cadherin)
  • monolayer of epithelium and endothelial formed, which ions cannot pass through so electric current is generated
  • film of liquid over epithelial layer to mimic surfactant of lungs
  • scientists measured integrity of layer by measuring resistance across the two membranes, comparing air-liquid interface to just liquid interface

air-liquid interface had faster increase in resistance and higher resistance, showing how the physiological air-liquid interface helps monolayer formation

system successfully applied stretch to the cells

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12
Q

how can lung on a chip be used to study lung inflammation?

A
  • when TNF is applied to epithelium, on endothelial side icam adhesion receptor is expressed for macrophage recruitment
  • when TNF is applied, neutrophils start to migrate from endothelial side to epithelial side
  • under E. coli infection of epithelium, icam receptors are expressed, neutrophils are recruited and migrate to epithelial side to engulf bacteria

lung on chip can mimic inflammatory response

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13
Q

what does uptake of nanoparticles require to get into tissues?

A

mechanical stretching:
- nanoparticles such as silica can induce icam on the endothelial layer, when there is strain/stretching
- leads to major icam adhesion expression
- need combination of stretching and silica for this response
- real world application for lung disease
- nanoparticles can reach blood flow and get into tissues – mimic lung disease

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14
Q

what is stiffness?

A

the stiffer something is, the more force is needed to make an indentation

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15
Q

what is the equation for stress?

A

stress = force/area
- σ = F/A
- N/m^2

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16
Q

what is the equation for strain?

A

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

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17
Q

what is the equation for stiffness?

A

stiffness = stress/strain
- E = σ/ε
- N/m2 = Pa

can use tangent of a stress/strain curve to find stiffness

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18
Q

what is shear stress?

A

stress that acts parallel to area

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19
Q

what is compression?

A

pushing force (N)

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20
Q

what is tension?

A

pulling force (N)
e.g. rubber band

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21
Q

how do different tissues have different stiffness?

A
  • Brain tissue is soft, 0.2kPa stiffness, breast is soft at 0.9kPa
  • Bone is very stiff at 30kPa
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22
Q

how can we mimic ECM stiffness in vitro?

A
  • Use polyacrylamide gels – their stiffness can be changed depending on the ratio of components
  • Polyacrylamide can go from soft (2kPa) to stiff (80kPa) to mimic ECM stiffness
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23
Q

what can ECM stiffness regulate?

A

stem cell differentiation
EMT
tumour progression

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24
Q

how does ECM stiffness regulate stem cell differentiation?

A

Depending on its physical environment, the stem cell decides what cell fate it will differentiate to:
Human MSCs on different ECM stiffnesses
- on soft, MSC differentiates to neurons
- On intermediate, MSC differentiates to muscle
- On stiff, MSC differentiates to bone

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25
Q

what is increased ECM stiffness diagnostic of?

A

diseased tissue:
- ECM/tissue stiffness correlates with disease
- e.g. fibrotic liver has much higher stiffness than normal liver

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26
Q

what can increased tissue stiffness assist?

A

Increased tissue stiffness can assist intervention and therapeutic decisions: biopsy, treatment
- e.g. as stiffness increases, chronic liver disease increases
- can test stiffness with elastogram MRI and then determine if patients need biopsy r not

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27
Q

what techniques can be used to measure cellular mechanical forces? at what range can they measure?

A
  1. atomic force microscopy: 5-10pN
  2. micropipette aspiration: 10pN-1nN
    - suck a piece of membrane into a tube - the more force needed shows higher membrane tension
  3. optical tweezers: 0.1-100pN
    - with light, you can move a sphere on a membrane and measure the force needed to move the particle on the membrane
  4. magnetic tweezers: 0.1-1nN
    - use magnetic field to move particle on membrane and measure force
  5. uniaxial stretcher: 1-20%
    - control force needed to stretch membrane e.g. vacuum of lung on a chip
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28
Q

what is atomic force microscopy?

A
  • Measures indentation at very small levels
  • Cantilever is pushed ontop of a cell
  • The more indentation the cantilever causes, the more the angle of a laser reflected from the top of the cantilever changes
  • Change in reflection angle can be measured to reflect the amount of indentation and force needed to push cantilever
  • Therefore can calculate stiffness (F/A)
29
Q

what is palpitation cancer diagnostics?

A

e.g. breast cancer self-examination
- patients feel a lump in their breast, as the tumour issue is stiffer than the surrounding breast tissue

30
Q

how can mechanical forces promote tumour progression?

A
  1. highly proliferative tumour cells generate lots of cells, meaning it increases in density
  2. tumours lead to more ECM secretion, so excess ECM contributes to stiffness
  3. cells such as fibroblasts surround the tumour with contractile activity, contributing to stiffening
  4. immune cells secrete cytokines, resulting in more ECM being secreted so more tumour stiffness
31
Q

what does change in ECM stiffness trigger in tumour progression?

A

Epithelial cells transform to mesenchymal cells (EMT) causes disruption in cell-cell contacts such as tight and adherens junctions, so cells rely more on ECM integrin interactions and start to migrate out of the primary tumour and can metastasise in the bloodstream

32
Q

how is tumour cell fate controlled by ECM mechanics?

A
  • Cells under high tension result from actin cytoskeleton squashing the nucleus
  • This changes the permeability of the nucleus via its nuclear pores, enabling oncogene factors such as YAP to enter the nucleus and affect gene expression
  • Change in ECM stiffness can alter signal transduction pathways of the cell
33
Q

how do the ECM and integrins affect tumour progression?

A

Matrix crosslinking forces tumour progression by enhancing integrin signalling
- Normal tissue is 0.2kPa, tumour tissue is over 1.5kPa
- 10-fold increase in stiffness

34
Q

what are the 3 main mechanosensors?

A
  1. Piezo channels
  2. integrins
  3. caveolae
35
Q

what is a piezo ion channel?

A
  • Piezo channels are mechanosensitive ion channels, mainly ion calcium channel
  • 38 TM domains
  • Integral membrane proteins connected to cytoskeleton
36
Q

how are Piezo channels activated?

A

Piezo is mechanically activated by membrane/cortical tension/stretch:
- Piezo channel opens when lipid bilayer is stretched
- The ion channel is connected to the actin cytoskeleton, so when the actin is pulled, the pore will also open
- If filamentous-actin is depleted, much higher force is needed to open the channel

37
Q

what are caveolae?

A

Caveolae means small caves: caveolae are small invaginations of the plasma membrane
- Similar to clathrin-coated pits
- Caveolin is the protein which activates the invagination
- Caveolae regulate membrane trafficking: caveolae-dependent endocytosis
- Caveolae can act as mechanosensors and sense stretch

38
Q

what is the composition of caveolae?

A
  • 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
39
Q

what causes caveolae to flatten?

A

caveolae flatten upon shear stress and increase in membrane tension

40
Q

how do caveolae function?

A

if cell is stretched and there is no extra membrane, the cell will tear
- caveolae act as a reservoir of extra membrane
- caveolae flatten out and provide extra membrane necessary to enable stretch of the cell
- under cell stretch, cavin disassociates from plasma membrane and acts as a signalling molecule to regulate transcription of genes
- biochemical changes are then induce

41
Q

what are integrins?

A
  • Integrins are major ECM stiffness sensors
  • They are transmembrane proteins which contact the ECM
  • Extracellular domain of integrin is normally inactive
42
Q

how are integrins mechanosensors?

A
  • When force is applied to the ECM or onto the integrin, there will be a clustering of integrins as a focal adhesion
  • Focal adhesions are interface between ECM and intracellular cytoskeleton – talin links integrin to F-actin (through vinculin)
  • F-actin is linked to vinculin, then to talin, then to the integrin
  • In vivo, focal adhesions mature into fibrillar adhesion, where talin is replaced with tensin
43
Q

how does talin act as a mechanosensor?

A
  • Talin is a multimodular molecule
  • When a force is exerted, talin can unfold
  • When talin is unfolded, vinculin can bind to a vinculin-binding site on talin to connect it to the actin cytoskeleton
  • This promotes the maturation of focal adhesion
44
Q

how is cell migration triggered via integrin-dependent adhesion and mechanotransduction pathways?

A
  • In cells, there is acto-myosin cytoskeleton connected to focal adhesions
  • Longer filaments are F-actin, and thicker filaments are myosin
  • Myosin motor proteins between the actin filaments exert force on the actin
  • Myosin slides the filaments against each other, which exerts a pulling force on the focal adhesions
  • Focal adhesions are connected to ECM, meaning the ECM provides a equal pushing force on the acto-myosin cytoskeleton
  • If the pulling force is greater than the pushing force, this leads to movement of the cell in the ECM
45
Q

what does stiffened ECM promote in cancer?

A

Stiffened ECM promotes epithelial-mesenchymal transition and invasion
- When ECM is stiffer, the structure of the epithelial cell clump is much flatter, and can migrate
- When ECM is soft, the structure is more compact and does not migrate
- When ECM is stiff, Snail protein is upregulated
- Snail is a key transcription factor of EMT
- Stiff ECM promotes Snail and therefore promotes EMT
- ECM stiffness contributes to tumour progression

46
Q

what inhibitors can prevent cell-ECM interactions to inhibit tumour progression?

A
  1. TGF-beta inhibitors: Pirfenidone, Fresolimumab
  2. Matrix metalloproteinase (MMP) inhibitors: Losartan
  3. LOXL2 inhibitors: Simtuzumab
  4. integrin inhibitors: antibodies
  5. Rho-kinase inhibitors: statin
  6. Focal adhesion kinase inhibitors: Defactinib
  7. Hyalouronic acid inhibitors: PEGPH20
47
Q

how are TGF-beta inhibitors important in preventing adhesion signalling?

A

TGF-beta inhibitors: Pirfenidone, Fresolimumab
- To inhibit the increase in ECM stiffness, inhibit TGF-beta growth factor which is known to upregulate collagen and fibronectin ECM molecules
- Counteract ECM production

48
Q

how are matrix metalloproteinase (MMP) inhibitors important in preventing adhesion signalling

A

MMP inhibitors (losartan) inhibit production of collagen and fibronectin, thus reducing stiffness
- block maturation of ECM

49
Q

how are LOXL2 inhibitors important in preventing adhesion signalling?

A

LOXL2 inhibitors (Simtuzumab) inhibit the LOXL2 enzyme which normally helps crosslinking of ECM fibres
- decreases the stiffness of ECM by inhibiting crosslinking activity

50
Q

how are integrin inhibitors important in preventing adhesion signalling?

A

Can target integrins with antibodies to interfere with integrin binding to ECM
- reduce cell-ECM interaction to prevent metastasis

51
Q

how are Rho-kinase inhibitors important in preventing adhesion signalling?

A

Can tackle downstream signal transduction via Rho-kinase inhibitors (statin) to block mechanotransduction pathway

52
Q

how are focal-adhesion kinase (FAK) inhibitors important in preventing adhesion signalling?

A

FAK inhibitors (Defactinib) prevent integrin downstream signalling and focal adhesion formation

53
Q

how are hyaluronic acid inhibitors important in preventing adhesion signalling?

A

Hyalouronic acid inhibitors (PEGPH20) interfere with post-translational modification of ECM

54
Q

what key oncogene is in the hippo pathway?

A
  • Excess YAP activation leads to organ enlargement e.g. eye in Drosophila
  • YAP may be involved in regulation of proliferation
  • YAP is an oncogene
55
Q

what regulates the YAP hippo pathway in mechanotransduction?

A
  • Mst1/2 kinase and Lats1/2 kinase both phosphorylate YAP/TAZ
  • YAP = Yorkie in Drosophila
  • Mammals have 2 subfamilies: YAP and TAZ
  • Lats1/2 phosphorylates YAP/TAZ at position 127 (Position 128 in yorkie)

When YAP is phosphorylated:
1. It can then be ubiquitinylated and then degraded
2. It can be bound to 14-3-3 molecule and prevented from entering the nucleus

Both ways prevent YAP entering nucleus and regulating transcription

56
Q

what regulates the hippo pathway?

A

Apicobasal polarity, mechanotransduction, cell-cell adhesion and contact inhibition regulate YAP

57
Q

what does the hippo pathway signalling control?

A

YAP regulates proliferation, cell survival, competition, stem cell maintenance, metastasis, regeneration

58
Q

what determines YAP nuclear localisation?

A

ECM stiffness

cell size

59
Q

how does ECM stiffness affect YAP nuclear localisation?

A

Cell at 0.7kPa (soft) and at 40kPa (stiff)
- Soft ECM cell has YAP excluded from nucleus
- Stiff ECM cell has YAP localised in nucleus
- In stiff ECM, downstream YAP molecules have been transcribed and translated – high YAP protein expression
- YAP is an oncogene, so stiff ECM promotes tumour progression

60
Q

how does cell size affect YAP nuclear localisation?

A

Geometry/cell shape determines subcellular localisation of YAP
- When YAP is in a small area, YAP is excluded from the nucleus
- When YAP is in a spread out, large cell, YAP localises in the nucleus

Spread-out cell on micropillars-coated in ECM, YAP is found in nucleus
- It is not the contact area that is important, but the geometry of the cell
- Spread out, large cell results in YAP in nucleus

61
Q

what regulates YAP nuclear localisation?

A

actin cytoskeleton and actin contractility:
- C3 Rho inhibitor prevents Rho-kinase activation of the cytoskeleton, resulting in YAP being excluded from the nucleus
- myosin inhibitor also prevents YAP being in the nucleus
- therefore cytoskeleton is needed to localise YAP in nucleus

62
Q

how does the actin cytoskeleton regulate YAP nuclear localisation?

A

Cells placed on micropillars, one pillar was rigid, the other was more flexible:
- When cell on rigid pillar, acto-myosin contractility is high, so YAP in nucleus
- When cell on soft pillar, acto-myosin contractility is decreased, so YAP is not in nucleus

63
Q

how is YAP important in stem cell differentiation?

A

YAP plays an important role in ECM stiffness-directed stem cell differentiation:
- MSCs on stiff (40kPa) matrix differentiate to bone cells (osteoblasts)
- When YAP is knocked down, MSCs on stiff matrix have decreased differentiation into osteoblasts
- YAP important in differentiation
- MSCs on soft (1kPa) matrix have changed differentiation to adipocytes
- If Rho is blocked, there is no differentiation as YAP cannot enter nucleus

64
Q

what is YAP?

A

a transcription regulator and an oncogene

65
Q

what key factors are involved in YAP nuclear localisation?

A
  • cell spreading/size, ECM stiffness and stiff pillars lead to nuclear accumulation of YAP
  • this causes cells to have high acto-myosin contractility
  • this leads to increased proliferation and MSC differentiation into osteoblasts
66
Q

what key factors are involved in YAP exclusion from the nucleus?

A
  • Soft ECM, confinement and soft pillars lead to exclusion of YAP from nucleus
  • this leads to apoptosis, growth arrest and promotion of differentiation into adipocytes
67
Q

what does soft ECM YAP-controlled MSC differentiation result in?

A

adipocyte formation

68
Q

what does stiff ECM YAP-controlled MSC differentiation result in?

A

osteoblast formation

Molecular cellular biology (13 decks)

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