mechanobiology Flashcards
what is mechanobiology?
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)
what are the four general steps in mechanobiology?
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
how does fluid flow effect cells?
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
give an example of the body detecting a mechanical stimuli and having a response? (x2)
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
how does the lung on a chip work?
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
how is the lung on the chip better than other in vitro models and how was this confirmed?
- 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)
- Air liquid interface better than just liquid
Made of PDMS - Applying the vacuum does stretch the cells (confirmed by experiments e.g. mapping same cell without vacuum onto cell with vacuum)
how was the lung on the chip used to investigate lung inflammation?
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
how did investigating reaction to silica (glass) nanoparticles show that replicating the mechanics of organs is important to get a realistic view?
Applied silica (glass) particles in the air channel of a lung-chip
Researcher looked at ICAM-1 (neutrophil receptor) expression in the epithelia
- Showed that immune response to these nanoparticles was minimal without the mechanical breathing action (stretching), but was much greater with it.
- Production of radical oxygen species also increased massively when the breathing/stretching AND the glass particles were applied together
- 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
what are the three (maths things) foundations of mechanical biology?
Shear = stress acting parallel to an area
Compression = pushing force (N)
Tension = pulling force (N)
what is the equation for stress
sigma = F/A
stress is just pressure, measured in N/m^2 or Pa (same thing)
what is the equation for strain?
little squiggly E thingy = change in length/ original length
unitiless (a length divided by another length)
give an idea of the stiffness in different tissues
Different tissues have different stiffness
e.g bone = 40-100KPa
brain =0.2KPa
what is the equation for stiffness?
E = stress or sigma / strain or squiggly E
as strain is unitless, stiffness is also measured in Pa or N/m^2
how can stiffness be emulated?
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
use MSC (stem cells) to explain how stiffness is important
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
what happens with stiffness when looking at fibrosis and how is this viewed?
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)
what is stiffness like in cancer for tumours and for healthy tissue?
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
why does cancer tissue have a higher stiffness than healthy tissue?
more cells packed closely together (density), cells secreting more ECM than normal, improved quality of cross links in the ECM
piezo channels - what are they?
An ion channel, 38 transmembrane domains
Directly responsive to stretch (lateral tension) pore opens like opening a door
what other theory exists to explain how Piezo channels work?
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
dysfunction of piezo channels is correlated to…
number of different diseases e.g. colorectal cancer (not fully understood so nothing confirmed)
integrins - what are they?
what is their structure like and how are they activated?
hint - disney pixar movie
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
what stimulus is communicated via integrins?
Changes in ECM stiffness communicated via integrins - to actin/myosin cytoskeleton - to nucleus via downstream signalling
more specifically, what is a focal adhesion and how do integrins work? hint - talin and Vi_
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
what are caveolae - appearance and function?
Means small cave
They appear as small invaginations of a plasma membrane
Caveolae allow the membrane to cope with lateral tension (stretching the membrane)
how do caveolae work?
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
what are stress fibres?
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
why might stress fibres be associated with the nucleus?
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
how does ECM stiffness effect Rho?
not just whaty the effect on Rho is but also how it occurs
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
explain three inhibitors that disrupt cell ECM interactions/adhesion signalling
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
what is the hippo pathway - what effects does it have and how is it regulated?
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)
explain the steps of the Hippo pathway
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…
what controls YAP subcellular localisation? how might this relate to cancer?
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
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
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
how is ECM stiffness related to cancer?
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
