Transcript Mechanical Force Effects on Cellular Functions

Mechanical Force Effects on
Cellular Functions
Kyle Wallenberg
Bioengineering 506
4/15/2009
Adhesion-mediated
mechanosensitivity: a time to
experiment, and a time to theorize
Alexander Bershadsky, Michael
Kozlov and Benjamin Geiger
Outline
• Introduction to mechanosensitive adhesion
structure/function
• Focal adhesions and focal complexes
• Focal adhesion mechanosensing adhesions
• Non-focal adhesion mechanosensing adhesions
• Mechanosensing signaling mechanisms
• Physical models
– Differences
• Conclusions
Introduction
• Adhesion-mediated signaling
– Provides cells with descriptive information about
microenvironment
– Mechanical influences
• Ability to grow and strengthen in response to applied
force
• Integrin-mediated cell matrix-adhesions
• Full understanding of mechanisms unknown
– Introduction of theoretical models to understand
adhesion-mediated mechanosensing
Adhesion Machinery
• Composed of specialized subcellular contact sites
– Formed by transmembrane receptors linked to
cytoskeleton
– Link cells to extracellular matrix (ECM)
• Few micrometers in length
• Sense chemical properties of external surfaces
• Respond to mechanical cues
– Mechanical forces (stresses)
– Mechanical deformations (strains)
Adhesion Site Functions
• Ensure correct and stable positioning of cells
• Unpredictable nature of external
perturbations
– Cell develops ‘just in time mechanism’
• Adhesion strength dependent on amount of
stress/strain applied at adhesion site
Dynamics of Mechanical Forces
• Strong forces can
disrupt local
adhesions
– Force-dependent
adhesion dissociation
• Cell-cell adhesions
• Cell-matrix adhesions
• Existence of forcedependent adhesion
growth
– This phenomena
intrigued scientists
Focal Adhesions
• Formed by different cell
types
– Fibroblasts
– Epithelial cells
• Large (several microns in
length)
• Adhere to actin filaments at
proximal end
• Formation dependent on
natural matrix proteins or
RGD peptides at high
densities
• Interact with ECM using
different integrins
Focal Adhesions (contd.)
• More than 100 proteins
make up FA domain
– Link integrin molecules and
actin filaments
– Regulate assembly and
signaling generated by
adhesive interactions
•
Gives FAs ability to
respond to range of
signals/features
– Rigidity
– Mechanical perturbation
– Topography
Focal Complexes (FXs)
• Type of cell-matrix
adhesion which FAs
evolve from
• Small (less than micron
in size)
• Formed continuously
from lamellipodia
• Some undergo
maturation
FX-to-FA Transition
• Dependent upon continually applied force
– (1) Inhibition of myosin II leads to accumulation of FXs and
disappearance of FAs
• Activation of myosin II induces FA assembly
– (2) Application of external force to FAs motivates growth in
force direction regardless of myosin II activation
– (3 )Size of FAs and force applied generally proportional
– (4) When strong enough forces unable to be generated,
large FAs not formed
• E.g. on a soft matrix
– Overall trend:
• FAs respond to local force by increased assembly
• FAs respond to relaxation of force by disassembly
FA Mechanosensing Adhesions
• Involved in many cellular responses
– Substrate stretching
– Variations in substrate rigidity
• Migration in direction of increasing rigidity
– Durotaxis: Directed movement of cell motility
– Fluid shear stress
Non-FA Mechanosensing Adhesions
• Integrin-mediated adhesion
– Fibrillar adhesions
• Associated with ECM fibrils
• Evolve from FAs in force-dependent manner
• Unlike FAs, do NOT disassemble when force relaxed
– Podosomes
• Different substrate rigidity response than FAs
– Lifespan, not shape, depends on substrate flexibility
Non-FA Mechanosensing Adhesions
• Cadherin-mediated cell-cell
adherens junctions (AJs)
– Depend on myosin-II driven
contractility
• Thus, the inhibition of myosin
II reduces AJ proteins at cellcell interface
• Platelet endothelial cell
adhesion molecule (PECAM)
– Immunoglobulin family
– Influences mechanical
response of endothelial cells
to fluid shear stress
Unanswered Questions Regarding FAs
• Relationship/interaction between FA components
and relative spatial organization mostly unknown
• Precise mechanisms by which cells respond to
environment unanswered
– Surface rigidity
– Ligand density
– Local/global mechanical perturbations
• Unknown which molecular interactions within
FAs regulated by force
Possible Mechanosensing Signaling
Mechanisms
• Modulation of
phosphorylationdependent proteinprotein interactions
– May explain forceinduced changes within
FA components
– FAK, Src, Fyn
• Protein tyrosine kinases
thought to be involved
Possible Mechanosensing Signaling
Mechanisms
• Adaptor Proteins
– Zyxin
• Recruitment to nascent FXs accompanies maturation
into FAs
• When force applied, translocates from FAs to stress
fibers following substrate stretching
• Adhesion-dependent pathways regulating
activities of Rho family GTPases and Rap1
• These mechanisms have yet to be integrated
into a specific molecular scheme
Physical Modeling
• Models proposed over the years to explain
events in adhesion-mediated
mechanosensitivity
– Physical mechanisms governing FAs
– Shear-stress profile along FAs
– Spatial distribution of FAs
– Effects of substrate elasticity on FA formation
Physical Modeling
• Three specific models of FA mechanosensing
described
– Stress-driven model
– Strain-driven model
– Thermodynamic model
• Models differ in assumptions concerning
physical factors underlying mechanosensing
and dynamic behavior of FAs during
growth/shrinkage
Stress-driven Model
•
Assumed that FAs contain molecular
switches that react to application of
force by changing states from inactive
to active, or vice-versa
– Sense stress and switch to active
conformation when stress exceeds a
critical value
•
Could be basis for explaining variety
of mechanisms involved in
mechanosensing:
– Stress-induced transition of certain FA
proteins
– Modulate activity of enzymes to turn on
or off phosphorylation switches
•
Kinases/ Phosphatases
– Regulate various adhesion molecules
•
•
•
Extracellular fibronectin
Adaptor proteins such as talin and
vinculin
Signaling enzymes such as Src and FAK
Stress-driven Model
• Mechanosensitive protein unit
connected to actin filament
moving with retrograde actin flow
– Results in dragging force acting on
protein unit from filament
• (a) – Passive state with weak slip
link
• (b) – Active state with strong slip
link
• Transition occurs when dragging
force exceeds critical value
– Leads to conformational change in
mechanosensitive protein
• Could explain mechanism for
maturation of FXs by ECM
elasticity
Strain-driven Model
• Mechanosenser switch activated
by local elastic strain
– Characterized by compression or
extension
• FA composed of two layers
– Lower layer contains
mechanosensitive proteins and
integrins and is attached to
substrate
– Upper layer connected to actin
filaments
• Transmits force to lower layer
• Compression of top of lower layer
relative to its bottom drives FA
assembly
– Generates strain at front edge of
FA leading to growth in pulling
force direction
Energy Considerations
• Stress-driven model
– Stress can promote conformational transition of
protein if thermodynamic work produced by
stress decreases activation energy of the
transition
• Requires significant change in protein dimensions
– Example is stretch-activated ion channels
• Open when 2-D membrane stress applied
• Change in area of stress-sensing channel in membrane
large enough to drive transition from passive to active
state
Energy Considerations
• Stress-driven model (contd.)
– Stress induced by actin filaments points in direction of
contractile or pulling force
– Conformational transition must result in protein stretching
(ΔL)
– Energy produced by stress is ΔF = -γ*L(per)* ΔL
• γ is lateral tension
• L(per) is protein linear dimension measured perpendicular to the
stress
– ΔL must be greater than 4 nm assuming stress-induced
force (γ*L(per)) acting on single protein is ~ 1pN and
energy produced by stress greater than thermal energy
– Points out limitation in sensing stress in case of FAs
Energy Considerations
• Strain-driven model
– Strain sensing understood if attachment of plaque
protein to mechanosensing layer coupled to
deformation of sensing layer
• If sensor layer not deformed prior to binding plaque protein,
energy of required deformation is paid at expense of binding
energy
• If sensor layer is deformed prior to binding, effective affinity
of plaque protein increases
• Why does this matter?
– Binding constant of plaque proteins can be altered by straining
mechanosensing layer
» Deformation energy coupled to binding of one plaque
protein must be larger than thermal energy
Thermodynamic Model
• No protein switch
• Elastic stress generated within plaque parallel to
plasma membrane by stress fibers can cause FA selfassembly and growth in pulling force direction
– Reducing pulling force leads to FA disassembly
• Elastic stress decreases chemical potential within
plaque, enhancing self-assembly by adding new plaque
proteins
• Predicts internal treadmilling-like motion of proteins
which can progress in different directions depending
on organization of FA assembly/disassembly
Thermodynamic Model
• (a) – FA composed of
aggregate of elastic building
blocks
– FA connected to substrate by
links along surface
• (b) – Pulling forces results in
aggregate stretching and
buildup of elastic energy
• (c) – FA assembly driven by
introduction of new
proteins into aggregate
– Concentration of new
proteins are distributed
diffusively throughout plaque
– Leads to energy relaxation
Implications of Thermodynamic Model
• FA plaque is elastic and can withstand mechanical
stress
• Cannot undergo stretch-induced rupture with
addition of new FA proteins
– May require molecular devices similar to formin
proteins
• Can maintain connection to protein complex while new
monomers are added to complex while at the same time
stabilizing the growing structure
• More modeling and experimentation necessary
to understand mechanism by which formins
direct FA mechanosensing
Differences Between Models
• Position of mechanosensor within the FA
– Stress-driven model
• Stress sensors located at interface between plaque and
stress fiber
– Strain-driven model
• Strain sensors located in integrin layer interacting with
ECM
– Thermodynamic model
• Elastic stresses of plaque stimulate FA self-assembly
– Effective mechanosensors located within plaque
Differences Between Models
• Proposed method of molecular exchange between FA
and cytoplasm
– Stress-driven model
• Stress-mediated transition stabilizes and reinforces connection
between stress fibers and plaque
– Results in FX maturation into FAs
– Strain-driven model
• Actin spread through the protein compresses mechanosensor
layer ahead of plaque and extends layer behind plaque
– Compressed molecular switches promote binding of new plaque proteins
while extended switches promote plaque disassembly at back of FA
» Results in FA treadmilling
– Thermodynamic model
• Involves entire plaque volume rather than partial areas along front
and back of FA (analogous to strain-driven model)
Conclusions
• Focal adhesion key example of a mechanosensitive adhesion
molecule
• Three FA mechanosensitive models introduced
– Stress-driven model
– Strain-driven model
– Thermodynamic model
• Adhesion-mediated mechanosensitivity described and confirmed,
but molecular mechanisms still not completely known
– Known that force promotes directional adhesion assembly
• Regulated by Rho GTPases and transformed by protein
phosphorlyation/dephosphorylation
– Unknown whether mechanosensitivity is directed by single protein or
complex nor what specific proteins are involved
References
• Bershadsky A, Kozlov M, Geiger B: Adhesionmediated mechanosensitivity: a time to
experiment, and a time to theorize. Current
Opinion in Cell Biology 2006, 18:472-481.
Questions