Mechanobiology Flashcards
What is mechanobiology?
The study of how physical forces and changes in cell or tissue mechanics contribute to development, physiology and disease
What is mechanotransduction?
The conversion of a physical force to a biochemical response (aka mechanosignalling)
What is mechanosensing?
When a protein or cellular structure responds to a physical cue to initiate mechanotransduction
What are the key steps in mechanotransduction? (4)
- Mechanosensing
- Signal transduction
- Signal integration at nucleus
- Cellular response
What is a physiological example of mechanotransduction? (3)
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
How does fluid flow affect the cytoskeleton of endothelial cells? (2)
- 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
How does the lung on chip work? (5)
- 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
What is a marker for tight junctions?
Occludin
What is a marker for cell adhesion?
E-cadherin
What is TER? (2)
- Transepithelial Electrical Resistance
- Lower resistance = tighter epithelial monolayer
How can lung on chip be used to study lung inflammation? (4)
- 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
How can lung on chip be used to study bacterial infection? (2)
- Apply E.coli to the epithelial layer
- Neutrophils adhere, migrate and engulf the E.coli on the epithelial side
How can lung on chip be used to study the effect of nanoparticles? (4)
- 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
What is the formula for stress?
σ = F/A
What is the symbol for stress?
σ
What are the units for stress?
N/m^2
What is shear stress?
Stress that acts parallel to an area
What is compression?
Pushing force (N)
What is tension?
Pulling force (N)
What is the formula for strain?
ε = ΔL/L0
What is the symbol for strain?
ε
What are the units for strain?
No units
What is the formula for stiffness?
E = σ/ε (stress/strain)
What is the symbol for stiffness?
E
What are the units for stiffness?
N/m^2 which = Pa
How can stiffness be studied in vitro?
Polyacrylamide gel can be made at different stiffnesses to mimic different ECM stiffnesses
How does ECM stiffness regulate stem cell differentiation? (4)
- 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
How is stiffness linked to disease? (3)
- 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
What is a hallmark of chronic liver and kidney disease?
Increased fibrosis (i.e. stiffness)
How can you measure stiffness? (6)
- Measure indentation of polyacrylamide
- Atomic force microscopy
- Micropipette aspiration
- Optical tweezers
- Magnetic tweezers
- Uniaxial stretcher
How is micropipette aspiration used to measure stiffness? (2)
- Suck a piece of membrane into a micropipette
- Higher stiffness = more force required
How are optical tweezers used to measure stiffness? (2)
- Move a small particle around inside the cell using light
- Higher stiffness = more force required to move the particle
How are magnetic tweezers used to measure stiffness? (2)
- Move a small particle around inside the cell using a magnetic field
- Higher stiffness = more force required to move the particle
How is a uniaxial stretcher used to measure stiffness? (3)
- Stretch the cell
- Measure how much force is required
- Higher stiffness = more force required
How is atomic force microscopy used to measure stiffness? (4)
- 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
Which factors contribute to tumour stiffness? (4)
- 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
How does mechanical stress affect tumour cell fate? (3)
- 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
What are the 3 main mechanosensors?
- Piezo channels
- Integrins
- Caveolae
How do piezo channels work as mechanosensors? (3)
- Mechanosensitive ion channels in the membrane
- Activated by stretch
- Connected to the actin cytoskeleton so also activated by pulling on the cytoskeleton
How do integrins work as mechanosensors? (5)
- 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
How do caveolae work as mechanosensors? (5)
- 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
How does stiffened ECM promote EMT? (2)
- Epithelial cells grown on stiff ECM upregulate snail
- Snail is a transcription factor which is important in EMT
What kinds of drugs can be used to inhibit cell-ECM interactions in cancer/fibrosis? (8)
-TGFβ inhibitors
- MMP inhibitors
- Losartan
- LOXL2 inhibitors
- Integrin inhibitors
- Rho inhibitors
- Hyaluronic acid (HA) inhibitors
- Focal adhesion kinase (FAK) inhibitors
Why are TGFβ inhibitors useful? (2)
- TGFβ promotes production of collagen and fibronectin
- Inhibitors reduce the amount of ECM that is secreted
Why are MMP inhibitors useful? (2)
- MMP is important for maturation and rearrangement of ECM
- Inhibitors interfere with the stiffness and amount of ECM available
Why are LOXL2 inhibitors useful? (2)
- LOXL promotes crosslinking of ECM
- Inhibitors reduce ECM stiffness
Why are HA inhibitors useful?
Interfere with post translational modification of the ECM
Why are FAK inhibitors useful?
Interfere with the formation of focal adhesions
Why are rho inhibitors useful? (2)
- Rho is important in actin filament production
- Interferes with downstream mechanotransduction signalling
How does the Hippo pathway work? (3)
- 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
How is the Hippo pathway regulated by mechanotransduction? (3)
- 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
Which experimental factors caused increased nuclear localisation of Yap? (3)
- Cell spreading
- Stiff ECM
- Stiff micropillars
All cause high actomyosin contractility
Which experimental factors caused exclusion of Yap from the nucleus? (3)
- Confined cell adhesion
- Soft ECM
- Flexible micropillars
All cause low actomyosin contractility
How does Yap influence differentiation? (2)
- 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
