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L2 Molecular Cell Biology > Mechanobiology > Flashcards

Mechanobiology Flashcards

1
Q

What is mechanobiology?

A

The study of how physical forces and changes in cell or tissue mechanics contribute to development, physiology and disease

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2
Q

What is mechanotransduction?

A

The conversion of a physical force to a biochemical response (aka mechanosignalling)

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3
Q

What is mechanosensing?

A

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

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4
Q

What are the key steps in mechanotransduction? (4)

A
  • Mechanosensing
  • Signal transduction
  • Signal integration at nucleus
  • Cellular response
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5
Q

What is a physiological example of mechanotransduction? (3)

A

Blood pressure autoregulation:
- Arterial diameter increases with increasing blood pressure
- A threshold is reached where Ca2+ is released which causes contraction of smooth muscle in the blood vessel
- Results in constriction to reduce blood flow and therefore blood pressure

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6
Q

How does fluid flow affect the cytoskeleton of endothelial cells? (2)

A
  • Fluid flow is recognised by the cell (e.g. by villi and cilia)
  • Actin filaments are organised in parallel so they align with the direction of the fluid flow
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7
Q

How does the lung on chip work? (5)

A
  • Breathing exerts mechanical stretch on the alveolar epithelium in the lungs
  • Chip is translucent for imaging
  • Epithelium grown in upper channel, endothelium grown in lower channel, separated by semi-permeable membrane
  • Epithelium exposed to air flow, endothelium exposed to fluid flow
  • Vacuum in the side chambers cause stretching of the cells which mimics breathing
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8
Q

What is a marker for tight junctions?

A

Occludin

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9
Q

What is a marker for cell adhesion?

A

E-cadherin

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10
Q

What is TER? (2)

A
  • Transepithelial Electrical Resistance
  • Lower resistance = tighter epithelial monolayer
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11
Q

How can lung on chip be used to study lung inflammation? (4)

A
  • Apply TNF to the epithelial layer
  • Causes expression of a cell adhesion factor
  • Apply neutrophils to the endothelial layer (flow freely in control conditions)
  • Neutrophils adhere and migrate to the epithelial side after TNF application
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12
Q

How can lung on chip be used to study bacterial infection? (2)

A
  • Apply E.coli to the epithelial layer
  • Neutrophils adhere, migrate and engulf the E.coli on the epithelial side
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13
Q

How can lung on chip be used to study the effect of nanoparticles? (4)

A
  • Breathe in nanoparticles which enter the lungs and migrate into the blood
  • Nanoparticles induce cell adhesion factors
  • The response is much larger when combined with stretching
  • The translocation of nanoparticles into the endothelial side is increased when stretch is applied
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14
Q

What is the formula for stress?

A

σ = F/A

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15
Q

What is the symbol for stress?

A

σ

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16
Q

What are the units for stress?

A

N/m^2

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17
Q

What is shear stress?

A

Stress that acts parallel to an area

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18
Q

What is compression?

A

Pushing force (N)

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19
Q

What is tension?

A

Pulling force (N)

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20
Q

What is the formula for strain?

A

ε = ΔL/L0

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21
Q

What is the symbol for strain?

A

ε

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22
Q

What are the units for strain?

A

No units

23
Q

What is the formula for stiffness?

A

E = σ/ε (stress/strain)

24
Q

What is the symbol for stiffness?

A

E

25
Q

What are the units for stiffness?

A

N/m^2 which = Pa

26
Q

How can stiffness be studied in vitro?

A

Polyacrylamide gel can be made at different stiffnesses to mimic different ECM stiffnesses

27
Q

How does ECM stiffness regulate stem cell differentiation? (4)

A
  • Grow stem cells on soft, intermediate and stiff ECM
  • Stem cells on soft ECM differentiate into neurons
  • Stem cells on intermediate ECM differentiate into muscle
  • Stem cells on stiff ECM differentiate into bone
28
Q

How is stiffness linked to disease? (3)

A
  • Fibrotic liver has significantly higher stiffness than normal liver when imaged using an elastogram
  • Stiffness increases with more advanced stages of chronic liver disease
  • Stiffness imaging can be used to diagnose/assess prognosis etc. rather than doing an invasive biopsy
29
Q

What is a hallmark of chronic liver and kidney disease?

A

Increased fibrosis (i.e. stiffness)

30
Q

How can you measure stiffness? (6)

A
  • Measure indentation of polyacrylamide
  • Atomic force microscopy
  • Micropipette aspiration
  • Optical tweezers
  • Magnetic tweezers
  • Uniaxial stretcher
31
Q

How is micropipette aspiration used to measure stiffness? (2)

A
  • Suck a piece of membrane into a micropipette
  • Higher stiffness = more force required
32
Q

How are optical tweezers used to measure stiffness? (2)

A
  • Move a small particle around inside the cell using light
  • Higher stiffness = more force required to move the particle
33
Q

How are magnetic tweezers used to measure stiffness? (2)

A
  • Move a small particle around inside the cell using a magnetic field
  • Higher stiffness = more force required to move the particle
34
Q

How is a uniaxial stretcher used to measure stiffness? (3)

A
  • Stretch the cell
  • Measure how much force is required
  • Higher stiffness = more force required
35
Q

How is atomic force microscopy used to measure stiffness? (4)

A
  • Cantilever pushes down on a cell to cause an indentation
  • Laser reflects off the cantilever
  • Angle of reflection changes with how much the cantilever indents into the cell
  • Measure the angle of reflection and how much force is being applied
36
Q

Which factors contribute to tumour stiffness? (4)

A
  • Cancer cells are highly proliferative so the tumour is denser than normal tissue
  • Excess ECM is secreted by cancer cells and is cross-linked
  • Cancer-associated fibroblasts surround the tumour and have a contractile structure which contributes to stiffness
  • Immune cells e.g. cytokines are attracted to the tumour and cause cancer cells to secrete more ECM
37
Q

How does mechanical stress affect tumour cell fate? (3)

A
  • EMT disrupts cell-cell adhesions in epithelial layer so the cell relies more on adhesions to the ECM
  • Cells are under low tension in an epithelial layer and under high tension as a single cell
  • Under high tension actin stress fibres change the permeability of the nucleus which causes changes in gene expression
38
Q

What are the 3 main mechanosensors?

A
  • Piezo channels
  • Integrins
  • Caveolae
39
Q

How do piezo channels work as mechanosensors? (3)

A
  • Mechanosensitive ion channels in the membrane
  • Activated by stretch
  • Connected to the actin cytoskeleton so also activated by pulling on the cytoskeleton
40
Q

How do integrins work as mechanosensors? (5)

A
  • Integrin is attached to the ECM and the actomyosin cytoskeleton via talin and vinculin
  • Applying force causes a cluster of integrins to form on the membrane called a focal adhesion in vitro
  • Talin can unfold under stress which exposes a binding site for vinculin, results in formation of focal adhesion
  • In vivo, fibrillar adhesions form with tensin instead of talin
  • Force generated by the actomyosin cytoskeleton is counteracted by the ECM exterting a force
41
Q

How do caveolae work as mechanosensors? (5)

A
  • Small invaginations of the plasma membrane which act as a reservoir of extra membrane to allow stretch
  • Contains caveolin and cavin membrane proteins
  • Membrane proteins are connected to the actin cytoskeleton
  • Pulling on the actin cytoskeleton flattens the invagination out
  • Cavin proteins can dissociate when there is stretch, regulate transcription
42
Q

How does stiffened ECM promote EMT? (2)

A
  • Epithelial cells grown on stiff ECM upregulate snail
  • Snail is a transcription factor which is important in EMT
43
Q

What kinds of drugs can be used to inhibit cell-ECM interactions in cancer/fibrosis? (8)

A

-TGFβ inhibitors
- MMP inhibitors
- Losartan
- LOXL2 inhibitors
- Integrin inhibitors
- Rho inhibitors
- Hyaluronic acid (HA) inhibitors
- Focal adhesion kinase (FAK) inhibitors

44
Q

Why are TGFβ inhibitors useful? (2)

A
  • TGFβ promotes production of collagen and fibronectin
  • Inhibitors reduce the amount of ECM that is secreted
45
Q

Why are MMP inhibitors useful? (2)

A
  • MMP is important for maturation and rearrangement of ECM
  • Inhibitors interfere with the stiffness and amount of ECM available
46
Q

Why are LOXL2 inhibitors useful? (2)

A
  • LOXL promotes crosslinking of ECM
  • Inhibitors reduce ECM stiffness
47
Q

Why are HA inhibitors useful?

A

Interfere with post translational modification of the ECM

48
Q

Why are FAK inhibitors useful?

A

Interfere with the formation of focal adhesions

49
Q

Why are rho inhibitors useful? (2)

A
  • Rho is important in actin filament production
  • Interferes with downstream mechanotransduction signalling
50
Q

How does the Hippo pathway work? (3)

A
  • Yap/Taz transcription factor enters the nucleus and promotes proliferation to increase organ size
  • Mst1/2 kinase phosphorylates Lats1/2 kinase to activate it
  • Lats1/2 kinase phosphorylates Yap/Taz which causes degradation/cytoplasmic retention of Yap/Taz so it can’t enter the nucleus
51
Q

How is the Hippo pathway regulated by mechanotransduction? (3)

A
  • Yap is localised in the nucleus at increased ECM stiffness but excluded at low stiffness
  • Cells grown in confined areas have low Yap nuclear localisation but cells which are allowed to spread have high Yap nuclear localisation
  • Cells grown on flexible pillars (lower actomyosin contractility, mimics soft ECM) have Yap excluded from the nucleus but rigid pillars cause Yap nuclear localisation
52
Q

Which experimental factors caused increased nuclear localisation of Yap? (3)

A
  • Cell spreading
  • Stiff ECM
  • Stiff micropillars
    All cause high actomyosin contractility
53
Q

Which experimental factors caused exclusion of Yap from the nucleus? (3)

A
  • Confined cell adhesion
  • Soft ECM
  • Flexible micropillars
    All cause low actomyosin contractility
54
Q

How does Yap influence differentiation? (2)

A
  • Cells which are under conditions that cause high actomyosin contractility have high Yap nuclear localisation and differentiate into osteoblasts
  • Cells which are under conditions that cause low actomyosin contractility have low Yap nuclear localisation and differentiate into adipocytes

L2 Molecular Cell Biology (13 decks)

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