Week 6 - Mechanotransduction & ECM biomimicry

Integrin mediated mechanotransduction

Amino acid sequence in ECM recognised by integren

Beta subinit of integrin recruits adhesion proteins (talin/vinculin) = focal adhesion complex

Focal adhesion complex works with actin filaments to generate forces on nuclear lamina (lamin A or A/C)

= can push/pull (loosen/condense) DNA = affects gene transcription

ECM properties

size/shape

protein composition

stiffness

(affects integrin mediated mechanotransduction)

• extracellular environment can influence traction force generation

Traction forces (actin-myosin)

Generated based on ECM stiffness

• sliding action between actin filament and heads of non-muscle myosin = traction force generated

• this movement of the myosin and actin filament allow the cell to move → much like the contraction of muscle

• Myosin 2 (non-muscle) is important for cell adhesion and migration

Focal adhesion complex

Protein complex that connects integrins to actin filaments

FAC proteins:

• Talin

• Vinculin

• Paxillin

• Focal adhesion kinase (FAK), and more

Talin-Vinculin binding sites & Mechanosensor properties

Talin & Vinculin can sense the strength of the tension due to their cryptic binding sites

• their binding sites are exposed/activated when the molecule is stretched

Talin = up to 9 binding sites for vinculin

Vinculin = up to 3 for MAPK

• concentration difference between bound and unbound vinculin can indicate the level of forces the cell is experiencing

• unbound = less force

Nuclear lamina

built by Lamin A/B/C and intermediate filaments

• higher conc. of Lamin A leads to greater stiffness

provides mechanical strength

• external environment can stiffen the nuclear lamina

Lines the nucleus to protect DNA

Stiff vs Soft ECM mechanotransduction

Soft = minimal integrins = minimal actin filaments = minimal traction force generated

Stiff = more traction force (opposite) = stiffer nucleus

YAP/TAX translocalisation

As stiff stiffness increases, expression of YAP/TAZ move from cytoplasm to nucleus

controls cell fate = differentiation

Larger traction force = translocalisation into nucleus = more transcription

• cell can react differently → either going into sleep or more activity

MRTFa forms

Similar to YAP/TAZ it acts as a co-transcription factor

1. MRTFa - G-actin complex:

   • cytoplasmic (inactive)

   • Globular form

2. MRTFa without G-actin:

   • when cell stiffens, g-actin is released → MRTFa enters nucleus → acts as transcription regulator

   • comes out of nucleus to bind with g-actin when stiffness decreases

Mechano-memory

if cells are exposed to high stiffness for longer period of time →

• sensitivity increased

• cell differentiation is prolonged

biomimicry process and challenge (3 steps)

1. Understand the normal conditions in healthy tissue → ECM in vitro

2. Mimic the disease → to study cell response

3. Cell death leaves scar tissues

ECM stiffness and tissue regeneration

stiffness is an obstacle in disease for regeneration

Stiffness can change due to:

• development stage

• ageing

• disease

Example (heart attack):

• after heart attack, stiffness increases

• causes stem cells to differentiate into bone (instead of cardiac muscle)

Hydrogel

water containing fibrous network

recapitulating few components of native tissue ECM

Stiffness controlled by

• altering amount of polymer

• and/or degree of crosslinking

Mimicking ECM properties (stiffness)

Step gradient = mimicking healthy tissue vs fibrotic cardiac tissue

• one strip of each

Linear gradient = mimicking linear gradient between healthy and fibrotic cardiac muscle tissue after MI

• gradual transition from healthy to disease-like

Durotaxis

stiffness driven cell migration

In the ECM:

• cells migrate to stiffer regions due to durotaxis

• cells sense stiffness via various integrin pairs (during durotaxis)

Can cause larger traction force on one side of cell

Digital stiffness writing

hydrogel polymerisation initiated by photo/thermo-initiation using either energy from photon or heat

• creating stiffens gel

• digital stiffness writing uses infared laser to cause increased stiffness in gel

Decellularised ECM properties and pros/cons

Sits on top of linear gradient PA gel

1. take out the native tissue and remove the cells

2. Freeze dry

3. Rehydrate to form ECM like hydrogel

Pro = better biomimicry compared to using one/few ECM components

Cons = hard to pinpoint main contributor to cell behaviour

Microcontact printing

micro-sized stamps that create an adhesive ECM island

• with specific shape and size to restrict/control cell shape/size

• can be specialised to also have different stiffness

Electrospinning

biomaterials can be electrospun to create random fibrous patterns

• mimic random arrangement of real tissue

3D bioprinting (biomimicry)

osteo-chondral interface can be mimicked by layer-by-layer 3D bioprinting

Chemically regulated biomaterials can...

alter stiffness + amino acid presentation

= control either/both stiffness and amount of integrin-binding ligands

= can examine cell mechanosensation