9/12/2013
Why and How Do Cells Sense
Mechanical Load
Biophysics Frontiers
9/11/2013
Outline
• Context of Cellular Biophysics /
Mechanobiology
• Story of Brenton Hoffman
– Interesting aspects of mechanotransduction
• Studying Mechanotransduction
• Development and use of a FRET-based
molecular tension sensor
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9/12/2013
What is a Cell?
• The basic functional units of life
– Variety of cell types with distinct components,
structures, and functions
– Comprised of numerous components and structures
• Topic of next couples classes
One Cell (fertilized egg)
Whole
bunch of
divisions
1013 specialized
cells (human)
Sea Urchin
Mouse
Seaweed
A Cell to Biologist / Chemist
“Cells are considered the basic
units of life in part because they
come in discrete and easily
recognizable packages. That's
because all cells are surrounded
by a structure called the cell
membrane.
http://www.nature.com/scitable/topicpage/what-is-a-cell-14023083
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9/12/2013
Corresponding Picture of a Human
Oxygen
We have a
characteristic
shape
A boundary (skin)
Carbon
Are made of
characteristic set of
elements
Hydogen
Nitrogen
Calcium
Pretty effective picture, when
you get sick, you get a pill
Phosphorous
Other
A More Complete Picture of the Cell
Skin: Plasma
Membrane
Skeleton and Muscles:
Actin and Myosin
Organs: ER,
Golgi, Nucleus…
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9/12/2013
Cell Migration: Cells generate forces
Human neutrophil chasing bacteria and avoiding red blood cells
Main Point
• Traditionally cell regarded as largely
biochemically-based system
– Bags full of chemicals
• Cells generate forces, so more to the story…
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9/12/2013
The Story of Brenton Hoffman
• 1997: Enrolled at Lehigh University
– To play football
– To study Chemical Engineering because, “I liked
chemistry in high school, but really don’t want to
spend all my time in a lab.”
• 2001: Enrolled at University of Pennsylvania
– Get PhD in Chemical Engineering
– Interview: “I really want to do something practical.
I think I will study the process control of industrial
bioreactors. That way I can get a nice job at
Merck.”
The Story of Brenton Hoffman
• First Week on campus, 2001: met Prof. John
Crocker, a Polymer Physicist study cell mechanics
• “I have no idea what this stuff is… This is
awesome.”
• Cell Mechanics
– Cells resist deformation
– Cells generate forces
– Cells “sense” mechanical variables
Generally, we don’t know how these processes work on
the molecular level
Very important in many disease states…
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9/12/2013
Cell are not like “Normal” Materials
Cells also generate forces, called active materials
Heart Cells – Cardiomyocyte
Courtesy of Nenad Bursac,
Duke BME
http://www.youtube.com/watch?v=SDWmVn6ScKI
Cellular Microrheology
Microrheology: the quantification of jiggle
Polymeric Material
∆r 2 (ω ) ∝
k BT
G * (ω )
Particle motion is determined by Brownian
motion and mechanical stiffness
Cell
∆r 2 (ω ) ∝
∆(ω )
G * (ω )
2
Particle motion is determined motor activity
and mechanical stiffness
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9/12/2013
Kinesin and dynein activity
Observe molecular processes with microscopy
Endogenous particles tracked with ~ 3 nm and 90 µs spatial resolution
Notice pulse-like temporal nature of the trafficking.
Observe 8 nm steps, with some larger steps, exactly as expected
The Story of Brenton Hoffman
• 2005: Department changes its name to
Chemical and Biomolecular Engineering
• 2007: Finish PhD in Chemical and
Biomolecular Engineering
– Want to be a Professor
– Need to do a Post-doc, but on what…
Apply knowledge of cell mechanics to
mechanotransduction
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9/12/2013
What is Mechanotransduction?
Mechanotransduction
Conversion of mechanical stimuli (applied forces, shear stress, or
extracellular rigidity) into biochemically detectable information
Simplified Schematic of Rho GTPase
regulation of actin structures
Force
Stress
?
Mechanical
Environment
Academically interesting b/c we don’t know how it works
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9/12/2013
Why study mechanotransduction?
Force sensing is important in many disease states
Atherogenesis
Laminar Flow
Atheroresistant
Disturbed Flow
Atheroprone
DeBakey, Annals of Surgery, 1985
Hahn, Nat Rev Mol Cell Bio, 2008
Why study mechanotransduction?
Force sensing is important in many disease states
Cancer
Anyone know the primary means of identifying breast cancer?
Butcher, Nat Rev Cancer, 2009
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9/12/2013
Why study mechanotransduction?
• Industrial Relevance
– Tissue Engineering
Manipulate cell
behavior by
controlling physical
environment
Mesenchymal stem cells plated on
different rigidity substrates
Engler, Cell, 2006
Why studying Mechanotransduction?
• Industrial Relevance: Tissue Engineering
– A major problem in vascular engineering creating
vessels that can withstand physical forces due to
blood flow and support proper cell function
• Current approaches copying the mechanical
environment of the embryo
– “Smooth muscles cells, which comprise vessel
wall, were stimulated with pulsatile radial stress at
165 beats/min and 5% radial distension to mimic
embryonic heart.”
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9/12/2013
Bioreactors with Mechanical Stimulation
Human muscle cells are attached to strands of collagen, or connective tissue.
They are then subjected to cyclic stretching (“exercise”) in a bioreactor, which
is a system designed to simulate the conditions of the human body. The preconditioning allows the cells to align in one direction, fuse to form muscle
bundles, and function like normal muscle.
http://www.wakehealth.edu/video/MultiMedia.aspx?id=38832
Story of Brenton Hoffman
• 2007: Starting Post-doc with Martin Schwartz,
Physical Chemist PhD who is a world renown
expert in Cell Biology and
Mechanotransduction
• Project: Make a molecular tension sensor
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9/12/2013
Now Begin More Formal
Mechanotransduction: Molecular Level
How can force effect molecules?
Conformational Change
Hoffman, Nature, 2011
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9/12/2013
Mechanotransduction: Cell Level
What cellular structures bear force?
Structural: Membrane and cytoskeleton
Adhesive: Focal Adhesions and Cell-cell contacts
Vascular Smooth Muscle Cell
Structures are also signaling centers
Hoffman, Nature, 2011
Mechanotransduction: Cell Level
Identified proteins that change activity level / localization
in response to mechanical stimuli, such as Src
Src Biosensor
Force applied with laser trap through bead
attached to integrins
w/o force
1 min force
5 min force
Wang, Nature, 2005
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9/12/2013
Mechanotransduction: Molecular Level
Talin Rod
Cryptic Vinculin
binding cites
Fluorescence Intensity
Vinculin
Time (sec)
Conversion of physical force into biochemical information
Del Rio, Science, 2009
Current Challenges
What is relationship between single molecule events and cell
behavior?
When and where are forces exerted across proteins in cells?
Are forces in cells large enough to cause conformation changes?
?
Need Molecular Tension Sensor
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9/12/2013
Outline
• Design of a Molecular Tension Sensor
– Picking the pieces
– Calibration
• Study of force mediated focal adhesion dynamics
– Molecular tension across vinculin mediates focal
adhesion assembly
• On going projects
Design Criteria
• Genetically encoded
• Applicable to multiple proteins
• Analog Signal
– Not just two state system
• Sensitive to spatio-temporal variations
– Reversible
• Compatible with standard microscopy
techniques
• Sensitive to pico-Newton forces
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9/12/2013
Picking the pieces: Technology
Forster Resonance Energy Transfer
Simple Picture
FRET is exquisitely distance sensitive
Picking the Pieces: Fluorophores
Strongest, brightest genetically encoded FRET pair
Argon Laser Lines
458 nm
514 nm
mTFP1 and Venus
Forster Distance = 6.0 nm
Cy3 – Cy5 = 6.0 nm
Wavelength (nm)
Day, J Biomed Optics, 2008
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9/12/2013
FRET Tension Sensor Concept
A calibrated spring between two fluorophores
would be a molecular tension sensor
FRET is inversely related to force
Picking the Pieces: Elastic Linker
Design Criteria
–
–
–
–
Extremely extensible
Extremely Reversible
Modular (Length Control)
Known / understandable mechanical
properties
Flagelliform
•
•
•
•
Main Component of spider silk
Based on a five amino acid
sequence
Weakly helical
Spider silk has been investigated
with AFM and simulations
– Responds to pN to nN forces
Becker, Nat Mat, 2003
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9/12/2013
Tension Sensing Module
o
o
o
o
Genetically encoded
Applicable to multiple proteins
Analog Signal
Sensitive to pico-Newton forces
Sensitive to spatio-temporal variations
Compatible with standard microscopy techniques
FRET Efficiency
Force Sensitivity
?
Force (pN)
• Set by linker composition and length and properties of
fluorophores
• Choose single molecule approach
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9/12/2013
TSMod Calibration
Empirical
Conversion
Analog Signal, Reversible, Force Sensitivity 1-6 pN
TJ Ha, Micheal Brenner, and Ruobo Zhou, University of Illinois
Grashoff, Hoffman, Nature, 2010
Tension Sensing Module
o
o
Genetically encoded
Applicable to multiple proteins
Analog Signal
Sensitive to pico-Newton forces
Sensitive to spatio-temporal variations
Compatible with standard microscopy techniques
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9/12/2013
Outline
• Design of a Molecular Tension Sensor
– Picking the pieces
– Calibration
• Study of force mediated focal adhesion dynamics
– Molecular tension across vinculin mediates focal
adhesion assembly
• On going project
– Next Generation Tension Sensors
Picking a Problem
Cell migration is a multi-step process involving force
generation and spatio-temporally regulated cell adhesion
Leading Edge
Trailing Edge
Important in many aspects of human health (wound healing, fighting infection…)
Parsons, Nat Rev Mol Cell Bio, 2010
human disease (metastatic cell migration,
atheroslcerotic development…)
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9/12/2013
Picking a Problem
Focal adhesions are mechano-sensitive sub-cellular
structures that mediate cell adhesion and
mechanotransduction
Leading
Edge
Trailing
Edge
Fluorescently labeled
focal adhesions
What is the molecular switch?
Picking a Protein: Vinculin
Cells must exert force to migrate. Forces are generated by F-actin
cytoskeleton and transmitted through focal adhesions (FAs) to the
extracellular matrix (ECM). Vinculin is a critical linkage in this process
Function: Molecular Clutch
Fast Actin Flow
Slow Actin Flow
Slow Cell
Movement
High Cell
Movement
Low Force
Transmission
High Force
Transmission
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9/12/2013
Vinculin Tension Sensor
Grashoff, Hoffman, Nature, 2010
Forces: FLIM-FRET of VinTS
Calibration
Average force across vinculin in stationary FAs is ~2.5 pN
With Maddy Parsons, King’s College, London
Grashoff, Hoffman, Nature, 2010
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9/12/2013
Vinculin Tension During Cell Migration
Individual
FAs
segmented
and
averaged
FRET Index
Migrating Bovine Aortic Endothelial Cells
Low molecular tension at back is surprising
Grashoff, Hoffman, Nature, 2010
Individual Focal Adhesion Dynamics
Vinculin -/- fibroblasts
reconstituted with
VinTS spreading on
fibronectin
t = 0 sec
Measured focal
adhesion dynamics
and molecular
tension
t = 870 sec
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9/12/2013
Average Focal Adhesion Dynamics
No Force
High Force
Suggests failure of vinculin to bear force is linked to disassembly
Grashoff, Hoffman, Nature, 2010
New Hypothesis
High Tension
Across Vinculin
Low Tension
Across Vinculin
Independent test: Vinculin should be required for increased
focal adhesion stability in response to applied force (focal
adhesion strengthening)
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9/12/2013
Focal Adhesion Stability
Vinculin -/- fibroblasts were transfected GFP-paxillin to mark FAs
Watched FA stability with and without vinculin in response to increased
force
RGB Overlays of 3 time points
White = stable FAs
Only with vinculin are stable adhesions achieved under increased
force
Grashoff, Hoffman, Nature, 2010
Force Across Vinculin Determines FA
Dynamics
Leading Edge
High Tension Across
Vinculin
Low Tension Across
Vinculin
Trailing Edge
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9/12/2013
Conclusions
Have created a FRET-based molecular tension sensor for vinculin
Genetically encoded
Applicable to multiple proteins
Analog Signal
Sensitive to spatio-temporal variations
Compatible with standard microscopy techniques
Sensitive to pico-Newton forces
Conclusions
• Revealed an unexpected regulatory mechanism in which the ability
of vinculin to bear force determines whether adhesions assemble
or disassemble under tension.
High Tension
Across Vinculin
Low Tension
Across Vinculin
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9/12/2013
The Story of Brenton Hoffman
• 2010: Publish this work in Nature
• 2011: Wrote the review of
Mechanotransduction you read
• 2012: Start at Duke as a BME Prof.
Lab Overview
• Determining force-sensitive
signaling pathways mediating
focal adhesion dynamics
– With tension sensor for the first
time can see where a protein is
under force.
• Mapping relationship between
protein tension and protein
dynamics
– Combining FRET and FRAP
• How are do molecular forces
across cellular adhesion
structures mediate collective
cell migration?
– How are sources of applied force
integrated?
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9/12/2013
Questions?
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