Lecture 26 Mechanobiology II Flashcards
A smaller change in length, when high stress is applied to a material means what?
The material is stiffer giving a higher value
What is the stiffness of plastic dishes relative to bone and brain?
Plastic dishes - very stiff = 100kPa
Bone - stiff
Brain - soft (0.3kPa)
What is the implication of the plastic dish stiffness?
Mechanically very non-physiological
How can tissue stiffness be mimicked?
Hydrogels - these can be tuned to different stiffnesses
How well do cells grow on hydrogel?
Not very well
What is added to the top of the hydrogel
A layer of ECM e.g. collagen or fibronectin to help the cells grow
What do ECM and tissue stiffness regulate
Stem cell differentiation
What differentiates into a range of cell types depending on the stiffness of the material
Human multipotent stem cells (MSCs)
What do MSCs differentiate into if grown on soft environment?
Neurons
What do MSCs differentiate into if grown on hard environment?
Bone
What do MSCs differentiate into if grown on intermediate environment?
Muscle
Where are MSCs exploited?
Stem cell therapy
How is stiffness and disease linked?
Where is this shown?
Increased stiffness can indicated and be used to diagnose diseased tissue
e.g. fibrotic liver is stiffer - clinician may palpate liver/abdomen
In chronic liver disease, what is there a correlation between?
Liver stiffness and stages of chronic liver disease
How is liver stiffness measured? Why?
Elastogram
- non invasive vs biopsy
- assesses patients prognosis and candidacy for treatment
- spare discomfort/risk complications of biopsy
How is breast cancer detected and why?
Self-examination
Since breast tissue is relatively soft (1kPa), tumours will be easily palpable, as they are stiffer than the surrounding environment.
What happens as a tumour proliferates?
- As a tumour proliferates, it exerts a mechanical force upon the surrounding tissue.
- Newton’s 3rd Law stipulates that this results in the same mechanical force being directed from surrounding tissue upon the tumour (for every action there is an equal and opposite reaction).
- This will change the properties of the ECM, making it stiffer than the tissue surrounding it. This occurs through changes to ECM secretions, amount of ECM secreted and cross-linking of ECM.
- It alters the signal transduction pathway and actin cytoskeleton by changing the actomyosin contractility of the surrounding cells, altering gene expression
- Interactions with the ECM is change as cell-cell adhesion is changed
- The epithelial monolayer is disturbed due to EMT
- Increased cell-ECM adhesion
Stiffness seems to contribute to what?
The change in stiffness seems to be part of the disease process and contributes to it’s progression, particularly to metastasis.
How does the ECM stiffness link to metastasis
The stiffer the ECM, the easier tumour metastasis will be.
What 5 techniques measure cellular mechanical forces?
- atomic force microscopy
- micropipette aspiration
- optical tweezers
- magnetic tweezers
- uniaxial stretcher
what does atomic force microscopy measure?
designed to measure local properties, such as height, friction, magnetism, with a probe
In atomic force microscopy, a cantilevered probe is used to indent the cell. A laser on-top of the cantilever will change the reflection angle of the laser beam (measured by a detector) to a degree which depends on the depth of the indentation. The identation is correlated to a specific force.
what does micropipette aspiration measure?
- Measures membrane tension by measuring the membrane potential (membrane clamping)
what are optical tweezers?
Use a highly focused laser beam to move a bead that will bind to the membrane to apply an attractive or repulsive force that to the cell/molecule
what do magnetic tweezers measure?
- Measure the tensile strength or the force generated by molecules
what do mechanosensors sense?
force
3 mechanosensor examples?
Piezo channels
Integrins
Caveolae
What are Piezo channels
Large ion channels (38 transmembrane domains)
What are Piezo channels directly activated by
Mechanical activity
There are the two ideas of how Piezo channels are activated. what is the first?
- When inserted into the plasma membrane, the channel will bend it. The application of a mechanical force to flatten the membrane will open the channel.
There are the two ideas of how Piezo channels are activated. what is the second?
- The channel sits laterally within the membrane. There is no bending. The application of a mechanical force - either directly to the channel or through the actin cytoskeleton being pulled, will open the channel.
How do we know actin plays a role in opening Piezo channels?
It has been shown that actin does play a role, as the loss of actin means that higher forces are needed to open the channel.
Describe the mechanosensingroles of Piezo1 in organisms
• Sensing endothelial fluid flow;
- If there is no flow, cells are disordered
- With flow, cells are ordered, including their actin cytoskeleton
• Regulating smooth muscle tone (inc. vascular tone);
• Altering red blood cell membrane tension through volume regulation
• Sensing lung tissue stretch to regulate breathing;
• Proprioception.
How do integrins work?
Integrins form dimers, and transduce conformational changes from the inside out (the opposite to usual when a ligand binds to a receptor), allowing interaction with the extracellular matrix.
What can integrins cluster into?
Focal adhesion complexes
What binds integrin to the actin cytoskeleton?
Talin - adaptor protein
What sort of molecule is Talin? What is the implication of this?
Mechanosensitive molecule - Talin can change its structure depending on the force applied to it.
What happens to Talin when a bigger force is applied?
It unfolds
How does Talin interact with F-actin?
Talin elongates and binds to F-actin. This change in conformation opens out further adhesion sites, exposing binding sites, onto which vinculin can bind.
When can Vinculin bind to Talin?
When talin is partially unfolded, otherwise the binding sites are occupied
What is necessary for Vinculin to bind to talin
When a force is applied
When do mature focal adhesions only form?
When force is applied
What contributes to the stabilisation of the focal point?
Vinculin
In summary, what does the maturation of focal adhesions depend upon?
Force application, unfolding of talin, binding of vinculin
What is Talin replaced by? What does this form?
Tensin
Fibrillar adhesion complexes
What are caveolae?
Stable membrane invaginations which are rich in Cavins 1-4, linked to the actin cytoskeleton/stress fibers via Cavin 1
What happens when shear stress/increase in membrane tension?
Caveolae flatten, dissociating cavin complexes and initiating signal transduction
Caveolae sense…
Stretch
