Lecture 9- Biophysics

Concepts of Mechanobiology

What are biological flows shape tissues and organs important for

inner ear and otolith formation, cardiovascular development, kidney morphogenesis and bone morphogenesis

What are some pathologies linked with abnormal flow cells interactions

embryonic heart and abnormal cilia

Mechanotransduction

Cells convert mechanical stimuli into electrochemical activity

Crucial for various physiological processes including touch, hearing, balance, and proprioception

Vital role in development, tissue repair, and the progression od diseasef

The majority of growth and remodelling of the vascular network takes place when

blood circulation has already initiated

What forces does flow velocity create

shear stress parallel to the tissue surface

What forces does pressure create

circumferential (tangential) and axial stress (along the long axis of the vessel)

What are potential flow mechanosensing complexes

flow sensitive membrane channels

caveolae

protruding cilia contain with mechanosensitive proteins

endothelial glycocalyx

What does glycocalyx experience

drag forces that are transmitted to the underlying cortical cytoskeleton as well as to distant integrin-dependent adhesions

Endothelial cell polarization, migration, differentiation

• Culture ECs elongate along flow direction

• However, ECs exposed to cyclic mechanical strain re-orient perpendicularly to the unidirectional shear stress generated by steady laminar flow

• Density dependant: high confluent cells migrate against the flow direction where low-density (isolated) cells migrate with the flow

• Endocardial cells: cells move towards-higher shear stress; endothelial mesenchymal transition leading valve formation

Why would you use a zebrafish to show the endothelial response to blood flow

• Transparent: Flow can be imaged in vivo

• Flow responsive genes can be image in vivo

• It can live without blood flow: flow can be perturbed

• in vivo

What are primary cilia, and how do they act as mechanosensors

Primary cilia are solitary, non-motile cilia found on most mammalian cells, projecting from the apical surface into the tissue lumen. They can detect mechanical stimuli such as:

• shear stress from fluid flow

• ECM distortion/compression in bone and cartilage

• biological flows in brain and spinal cavities

When bent by these forces, they can trigger ionic fluxes, especially Ca²+ signalling, which can then alter cell behaviour such as angiogenesis.

What happens to endothelial cilia in zebrafish embryos

• Endothelial cilia are present during angiogenesis and deflected by low flow forces

• Cilia deflection leads to endothelial calcium increase as flow forces increase

valvulogenesis

Complex embryonic process of developing heart valves from gelatinous endocardial cushions into think, functional fibrous leaflets (flow dependent)

conservation of momentum eqn

delta p = u delta² u

conservation of mass

delta u =0

boundary conditions from wall dynamics

u|omega = ub

What shows tissue convergence is flow dependent

endocardial cells converge toward the area of high shear and oscillatory flow; not exactly the direction of net flow

How does direct imaging of mechanotransduction work

• Shear stress is perturbed exogenously by injecting a bead

• Calcium imaging using GcaMP reporters, confirms shear stress mechanotransduction

What are the main stages of cardiac valve development in the zebrafish atrioventricular canal (AVC)?

Cardiac valve development in the atrioventricular canal (AVC) happens in a sequence:

• 28–48 hpf: cellular volume regulation

• 54 hpf: cell protrusion and EndoMT (endothelial-to-mesenchymal transition) begin

• 72 hpf: VIC formation and delamination

• 126 hpf: elongation and cardiac cushion remodelling

Cell types involved

• EdCs = endothelial/endocardial cells

• MCs = myocardial cells

• VICs = valve interstitial cells

• ECM = extracellular matrix

how are the mechanical forces from oscillatory blood flow come from

detected by some sensory machinery

What do ECs experience a broad range of mechanical stimuli…

• different flow profiles

• shear stress magnitude and gradients

• direction

• temporal gradients and frequency content

How does circumferential stress regulate endothelial-to-hematopoietic transition (EHT) in the dorsal aorta?

During zebrafish development, blood flow generates circumferential stress in the dorsal aorta (DA), especially on the ventral side. This mechanical deformation helps drive endothelial-to-hematopoietic transition (EHT), where endothelial cells become hematopoietic stem/progenitor cells.

Key points

• EHT occurs from cells in the ventral wall of the dorsal aorta

• Pulsatile blood flow causes the DA to deform strongly

• This creates tissue stress, greatest at the ventral side

• Endothelial cells move toward the ventral side

• Stem/progenitor cells are then extruded from the endothelium

• This extrusion depends on actomyosin contraction

How do cell–ECM and cell–cell interactions mediate mechanotransduction?

Mechanotransduction occurs through both cell–ECM adhesions and cell–cell junctions, which allow cells to sense, transmit, and respond to forces.

Cell–ECM interactions

• Cells attach to the extracellular matrix (ECM) through integrins

• Integrins connect to focal adhesions (FAs)

• FAs contain mechanosensitive proteins that transmit force to the cytoskeleton

Cell–cell interactions

• Neighboring cells are linked by gap, adherens, tight, and desmosomal junctions

• These junctions connect to the cytoskeleton

• They help distribute forces across tissues and regulate collective migration and junction remodelling

• Key mechanosensitive proteins

  • talin

  • vinculin

  • myosin-II

  • Anillin may also help transmit tensile forces through vinculin recruitment

  Additional signaling

  • Under mechanical stress, ATP can be released through Pannexin-1

  • ATP then activates purinergic receptors in neighboring cells

  • This helps regulate cell–cell tension

gap junctions

these are clusters of channels that form tunnels of aqueous connectivity between cells. They allow ions and small molecules to pass freely between cells, facilitating communication and coordination

adherens junctions

connect the actin filaments of neighbouring cells

tight junctions

seal between adjacent cells, preventing the passage of molecules and ions, maintain the selective permeability of epithelial layers

Desomosomes

strong connections that join the intermediate filaments of neighbouring cells

What is an example of measurement and manipulation of mechanical cues

cantilever-based systems, traction force microscopy, laser ablation, micro aspiration, approaches to stretch or compress tissues, strain mapping

Cantilever-based systems

• some of the most common methods in measuring tissue mechanical properties

• cantilevers as force-transducers which are key for indenting or deforming small regions of tissue

• contact forces and depth of indentation or tissue strain are recorded and can be used to calculate a modulus

• commercial atomic force microscopy (AFM) systems are cantilever-based systems that were first adopted for in vitro studies of cell monolayers

• AFM can report mechanical properties from 1 to 5um of the surface

Example of role of mechanics in pathfinding

• neurite outgrowth of cultured RGCs was found to respond to substrate stiffness, which depends on the stretch-activated ion channel

• the study showed that RGC axons sense a stiffness gradient in the brain and grow towards softer tissue

Microaspiration

Microaspiration involves applying pressure to pull a tissue into a narrow channel. The modulus or compliance of cells on an embryo or compliance of a patch of cells on an embryo or aggregate can be calculated from the geometry of the channel, the pressure applied, and the distance the tissue moves into the channel.

Optical tweezers velocimetry

• optical tweezing experiments to characterize blood cell motion

• detection of the constrained blood cell motion within the optical tweezer reflects the effects of surrounding flow forces

• can be sued for flow velocimetry by measuring displacement from the centre of the optical trap

• requires optical access, and low drag forces

traction force microscopy

• the traction force applied by the cell is measured from the deformation of the substrate

• substrate strain inferred by movements of embedded microbeads/nanobeads or displacement of micropatterns

laser ablation

• tension measured as relaxation time following ablation

• estimate of the properties requires fitting a viscoelastic model of the tissue

• invasive measurement

optogenetics - How is optogenetics used to control cell contractility through RhoA?

Optogenetics can control cell mechanics by using light-sensitive proteins to switch RhoA signaling on or off.

Example system

• A RhoA activator (the DHPH domain of ARHGEF11) is fused to the light-sensitive protein CRY2 → optoGEF-RhoA

• Light causes CRY2 to change conformation and bind its partner CIBN

What happens after illumination?

CIBN can be targeted to different places:

• Membrane-targeted CIBN
  → recruits optoGEF-RhoA to the membrane
  → activates RhoA
  → increases actomyosin contractility

• Mitochondria-targeted CIBN
  → pulls optoGEF-RhoA away from the membrane
  → reduces RhoA signaling at the cortex
  → decreases contractility

ferrofluid microdroplets

• Magnetic field makes droplets elliptic

• strain ɛ is obtained from the droplet′s aspect ratio, b/a

• Fitting a model provides info on mechanical properties

Mechanical properties can be

extrinsic or intrinsic

What is an important contribution to tissue and organ development

biomechanics of the microenvironment

Mechanotransduction contributes to…

cell orientation, migration and differentiation

Many new tools are available to…

measure the mechanical properties and perturb the micromechanical environment, and measure cellular response to mechanical cues

What is needed to fully understand the biomechanics of the microenvironment and causal effects with the cellular response

A combination of mechanical modelling and image analysis

biomechanics

mechanical processes that directly shape living organisms. Genes and environment are involved in directing material properties

compliance

the ability to deform under an applied force

compression

the object is under compression when it experiences a negative strain

elastic modulus

defines the elastic behaviour under an applied stress

force

an interaction with a magnitude and direction that changes the motion of an object or deforms it

mechanobiology

the feedbacks from mechanical processes that guide biology

tension

experiencing a positive strain

stiffness

the resistance to deformation under an applied force

strain

a dimensionless term to describe the deformation of an object caused by the force applied

stress

amount of force that is applied to a unit area

viscoelasticity

combination of viscous behaviours and elastic behaviours