mechanobiology

Question 1

what is mechanobiology?

Answer 1

the study of how tissue forces and changes in cell or tissue mechanics contribute to development, physiology and disease(how physical forces change cellular behaviour)

Question 2

what are the four general steps in mechanobiology?

Answer 2

Mechanosensing = when a protein or cellular structure responds to a physical cue to initiate mechanotransduction

Mechanotransduction = conversion of physical force to biochemical response. Transduction often along the cytoskeleton

Integration at nucleus = often transduction of signals accumulate at the nucleus to potentially regulate gene transcription/nuclear pore opening

Response = can me microseconds to minutes, affecting cell shape, motility, growth and fate

Question 3

how does fluid flow effect cells?

Answer 3

Changes in cytoskeleton upon fluid flow - looking at actin in endothelial cells. Applying fluid flow causes actin to align with direction of fluid flow to counteract the force of the fluid flow

Question 4

give an example of the body detecting a mechanical stimuli and having a response? (x2)

Answer 4

stereocilia and regulation of ion channels

blood pressure autoregulation - an inc. in bp detected in arteries resulting in Ca2+ release and vasoconstriction to reduce blood flow at a certain blood pressure threshold

Question 5

how does the lung on a chip work?

Answer 5

Based on microfluidics, has a tiny channel through which fluid flows, and also a channel for airflow to mimic expansion and contraction

Recreates the mechanical conditions as well. Set up like the lungs in layers - airflow - epithelium - membrane - endothelium - fluid flow. Vacuums either side can be induced and released to mimic inflation and deflation

Question 6

how is the lung on the chip better than other in vitro models and how was this confirmed?

Answer 6

1. forms proper monolayers and tight junctions (a key function of epithelial layers - boundaries). This was confirmed by measuring trans-epithelial resistance (the higher the better)
2. Air liquid interface better than just liquid
   Made of PDMS
3. Applying the vacuum does stretch the cells (confirmed by experiments e.g. mapping same cell without vacuum onto cell with vacuum)

Question 7

how was the lung on the chip used to investigate lung inflammation?

Answer 7

Applied TNF (tumour necrosis factor) to mimic inflammation in the epithelium layer

Observed endothelium - an adhesion receptor not expressed in the control group was suddenly expressed in treatment group, it binds to neutrophils from the ‘blood’

Neutrophils can then make their way to the epithelium layer in minutes

note - (Repeated similar experiment by adding E. coli instead of TNF):
E. coli causes secretion of TNF from the epithelial cells - expression of adhesion receptor in endothelium - neutrophils bind and migrate to the epithelium - phagocytose the bacteria

Question 8

how did investigating reaction to silica (glass) nanoparticles show that replicating the mechanics of organs is important to get a realistic view?

Answer 8

Applied silica (glass) particles in the air channel of a lung-chip
Researcher looked at ICAM-1 (neutrophil receptor) expression in the epithelia

1. Showed that immune response to these nanoparticles was minimal without the mechanical breathing action (stretching), but was much greater with it.
2. Production of radical oxygen species also increased massively when the breathing/stretching AND the glass particles were applied together
3. With the stretching, the nanoparticles were seen to travel into the blood (endothelial) channel from which they could theoretically travel to other organs in vivo, but this was not seen without the stretching

Question 9

what are the three (maths things) foundations of mechanical biology?

Answer 9

Shear = stress acting parallel to an area
Compression = pushing force (N)
Tension = pulling force (N)

Question 10

what is the equation for stress

Answer 10

sigma = F/A

stress is just pressure, measured in N/m^2 or Pa (same thing)

Question 11

what is the equation for strain?

Answer 11

little squiggly E thingy = change in length/ original length

unitiless (a length divided by another length)

Question 12

give an idea of the stiffness in different tissues

Answer 12

Different tissues have different stiffness

e.g bone = 40-100KPa
brain =0.2KPa

Question 12

what is the equation for stiffness?

Answer 12

E = stress or sigma / strain or squiggly E

as strain is unitless, stiffness is also measured in Pa or N/m^2

Question 13

how can stiffness be emulated?

Answer 13

Emulating stiffness -
Polyacrylamide can be used to create different stiffnesses (by altering ratios of the acrylamide and bis-acrylamide) then adding a thin collagen layer to allow cells to attach

note - it is clear = suitable for microscopy

Question 14

use MSC (stem cells) to explain how stiffness is important

Answer 14

MSC (stem cells) can either become muscle, bone or neuronal cells
The stiffness of the environment informs stem cells how to differentiate

Soft conditions = neurons more likely/preferential choice
Medium = muscle
Stiffest = bone cells

Question 15

what happens with stiffness when looking at fibrosis and how is this viewed?

Answer 15

Organs can become ‘stiff’ = fibrosis

Ultrasound can be used to measure this in an ‘elastogram’, a non-invasive technique that can assist in defining the stage of liver disease and avoid biopsy (how long the waves take to pass through and return, so take longer to pass through harder materials)

Question 16

what is stiffness like in cancer for tumours and for healthy tissue?

Answer 16

tumour has higher stiffness than the healthy tissue

more specifically, premalignant tissue has higher stiffness than healthy tissue, then even higher is malignant tumour, which can actually cause adjacent healthy tissue to have a slight increase in stiffness by exerting a force on it

Question 17

why does cancer tissue have a higher stiffness than healthy tissue?

Answer 17

more cells packed closely together (density), cells secreting more ECM than normal, improved quality of cross links in the ECM

Question 18

piezo channels - what are they?

Answer 18

An ion channel, 38 transmembrane domains
Directly responsive to stretch (lateral tension) pore opens like opening a door

Question 19

what other theory exists to explain how Piezo channels work?

Answer 19

not exactly directly responsive, instead connected actin filaments respond to mechanical stimuli, with the myosin motor pulling the actin causing opening of the piezo pore

Question 20

dysfunction of piezo channels is correlated to…

Answer 20

number of different diseases e.g. colorectal cancer (not fully understood so nothing confirmed)

Question 21

integrins - what are they?

what is their structure like and how are they activated?

hint - disney pixar movie

Answer 21

Transmembrane receptors that link cells to ECM

structure = Heterodimers with an alpha and beta subunit. These look sad - heads hanging down = inactive

activation = phosphorylation by focal adhesion kinase of the cytosolic domain, causing ‘inside out’ signalling, changing the extracellular domain so that it can engage with the ECM

Question 22

what stimulus is communicated via integrins?

Answer 22

Changes in ECM stiffness communicated via integrins - to actin/myosin cytoskeleton - to nucleus via downstream signalling

Question 23

more specifically, what is a focal adhesion and how do integrins work? hint - talin and Vi_

Answer 23

Focal adhesion is the oligomerisation of integrins with talin (talin is what connects integrins to the cytoskeleton, while there other end is linked to the ECM)

Talin - bound to F actin in the cytosol on one end, then to integrin on the other end. If force applied, talin can partially unfold certain domains, generating binding site for something called vinculin
Vinculin stabilises the interaction with the F-actin

Question 24

what are caveolae - appearance and function?

Answer 24

Means small cave
They appear as small invaginations of a plasma membrane
Caveolae allow the membrane to cope with lateral tension (stretching the membrane)

Question 25

how do caveolae work?

Answer 25

Cav1 is a protein present that interacts with stress fibres in the cell
Cavin molecules/complex are also present, attached to caveolae, involved in signal transduction…

Principle - flattening of the caveolae upon lateral tension, cavin complex detach (from CAV1? IDK) and cause downstream signalling

Question 26

what are stress fibres?

Answer 26

contractile structures composed of actin filaments (F-actin) and myosin II motor proteins. At least one anchor point for stress fibres are focal adhesions

they can be connected to the nucleus

Question 27

why might stress fibres be associated with the nucleus?

Answer 27

usually connected to the nucleus by nesprins and SUN proteins

helps maintain cell shape

method of mechanotransduction - getting mechanical signals to influence things like gene expression in the nucleus

Question 28

how does ECM stiffness effect Rho?

not just whaty the effect on Rho is but also how it occurs

Answer 28

Increase in ECM stiffness activates Rho pathway via integrins -

Rho is a small GTPase:
Activates YAP/TAZ (more later)

Rho also activates ROCK - Rho dependent kinase,

which phosphorylates MLCP (myosin light chain phosphatase) and stops it,

increasing MLC phosphorylation,

increasing contractility /motility in the cell

Question 28

explain three inhibitors that disrupt cell ECM interactions/adhesion signalling

Answer 28

LOXL2 (lucile oxidase like 2) inhibitors - prevents LOXL2 enzymes form cross linking in ECM, preventing increases in stiffness/fibrosis

Integrin inhibitors - antibodies inhibiting interaction of integrins with the ECM

Rho inhibitors - myosin light chain phosphorylation reduced, so contractility is prevented

Question 29

what is the hippo pathway - what effects does it have and how is it regulated?

Answer 29

Originally identified as a pathway regulating the size of organs

controls proliferation, survival, metastasis, regeneration, cell competition

regulated by mechanotransduction as well as cell-cell adhesion and contact inhibition (and apicobasal polarity)

Question 30

explain the steps of the Hippo pathway

Answer 30

YAP/TAZ is a transcriptional regulator (not directly interacting with DNA, but instead regulates the activity of transcription factors)

Membrane regulators activates Mst1 — a kinase so it phosphorylates Lats1 —- another kinase, phosphorylates YAP/TAZ at S127

Phosphorylation can cause YAP to be degraded, or retained in the cytosol by a protein called 14-3-3, where it can have no effect

These two results prevent YAP from entering the nucleus and causing its effects - proliferation, survival, metastasis, regeneration, cell competition etc…

Question 31

what controls YAP subcellular localisation? how might this relate to cancer?

Answer 31

the stiffness of the ECM:

Immunofluorescence labelling showed stiff ECM caused YAP localisation in the nucleus (where it can cause all kinds of effects like proliferation and metastasis), vs soft ECM causing YAP localisation in the cytosol

Question 32

more YAP is seen in the nucleus when the cell occupies a larger area.

Explain the experiments that proved cell size has this effect, and that it is not just area of contact with the ECM

Answer 32

Used cells grown on micropillars coated in ECM (so the cells are the same size, but only small areas of the cell were in contact with the ECM)

to compare to cells grown on regular ECM (large area of contact). These large cells had the same level of nuclear localisation of YAP for the two groups,
whereas smaller sized cells had much lower levels of YAP in the nucleus.

This tells us it is not the amount of cell ECM contact, but the size of the cell that affects YAP localisation

Question 33

how is ECM stiffness related to cancer?

Answer 33

EMT - epithelial to mesenchymal transition, a common step in cancer
Epithelial cells dedifferentiate

Structure loses it’s order/less spherical and more random

This is promoted by a stiffer ECM