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Mechanotransduction:
Examples
Eric Salm
BIOE 506
April 15, 2009
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Mechanotransduction: Recap
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Means by which cells convert a
mechanical stimulus to some chemical
activity
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http://www.nature.com/nrm/journal/v10/n1/pdf/nrm2605.pdf
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Article: Local Action
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Forced Unfolding of Proteins within Cells
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Authors:
Colin P. Johnson, Hsin-Yao Tang, Christine
Carag, David w. Speicher, Dennis E. Discher
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Publishing Journal: Science (2007)
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Outline
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Goal
Introduction
Methods
Results
Discussion
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Goal
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Identify cytoskeletal proteins that
change conformation due to
physiological stresses
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Introduction
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Protein domain unfolding has been
shown to occur; however, limited to
single-molecule measurements
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Introduction (2)
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Moving beyond single molecule to live
cells
Cys-”shotgun” method used
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Cysteine is a hydrophobic AA which results
in frequent AA-shielding
As a protein deforms, previously shielded
cysteines can now be labeled
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Introduction (2)
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Interested in membrane bound proteins
Spectrin (RBC): Two chains, α and β, cross-link F-actin.
Found to unfold at low forces. Central role in cell
deformability.
Myosin IIa (MSC): Responsible for actin-based motility
Vimentin (MSC): Member of intermediate filament
family of proteins. Intermediate filaments play an
important role in cell structure
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Methods: RBC analysis
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1a. RBCs reversibly lysed to make hemoglobindepleted cells
2a. Cys-reactive fluorophore (IAEDANS) added to
the cells
3a. Resealed entraping the fluorophore
4a. Ranged of tests from static to stressed
5a. Cells relysed releasing unbound fluorophore
6a. Cells denatured and all Cys that weren’t dyelabeled were alkylated with iodoacetamide (IAM)
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Results
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Analysed with separation of proteins by
SDS-PAGE gel electrophoresis
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Results (2)
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Analyzed using Liquid Chromatography-coupled
tandem Mass Spectrometry (LC-MS/MS)
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Sequential Two-Dye Labeling
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1. Static-accessible sites labeled to
near saturation with IAEDANS
2. Shear applied with labeling by a
secondary cys-flurophore (BODIPY-IA)
Done to magnify the difference
between shear and static Cys-labeling
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Fits force-dependent
Linderstrom-Lang
(fLL) scheme
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Shear stress,
proportional to force,
shifts the
confirmational
equilibrium
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Methods: Mesenchymal Stem Cells
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1. Membrane-permeable, Cys-labeling
fluorophore monobromobimane used to
label both tensed and relaxed cells
2. Cell lysates Cys-quenched
3. SDS-PAGE run
4. LC-MS/MS run
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Tensed vs Relaxed
(Blebbistantin)
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Results
Nonmuscle Myosin IIA
Vimentin
↑
→
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Conclusions
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Cys-labeling provided evidence of forceinduced changes within the structure of
cellular membrane proteins
Limited because it does not touch on
conformational changes in real-time
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Articles: Deep Cytoplasm
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Rapid Signal Transduction in Living Cells Is a
Unique Feature of Mechanotransduction.
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Authors:Singsoo Na,…, Yingxiao Wang
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Publishing Journal: Proceedings of the National
Academy of Sciences (May 2008)
Visualizing the Mechanical Activation of Src
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Authors: Yingxiao Wang et al.
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Publishing Journal: Nature (April 2005)
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Goal
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Pick up where last paper left off…
Move into real-time, live cell imaging
Examine the effects of stress within the
cell in terms of stress-induced Src
activation
Determine pathway for deep
cytoplasmic Src activation
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Src Background
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Tyrosine Kinase
First TK to be discovered
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Linked with Rous sarcoma virus
Served to link virus with cancer through
gene expression
Involved in intracellular signalling
Oncogene
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Activating Src-Type Protein
Use FRET to measure
activation of Src by use of
a FRET-based Src reporter
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Measures CFP vs YFP.
Enhanced CFP = active Src
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Methods:
Stress-Induced Src Activation
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1. Transfect CFP-YFP cytosolic Src reporter
into SMCs
2. RGD-coated magnetic bead was then
bound to integrin receptors on the cell
membrane
3. Local stress induced by turning on a
magnetic field
4. Quantify changes in Src activities by using
FRET ratios of the Src reporters in the
membrane
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Rapid Src activation: Integrin
-Stress
+Stess, + 0.3 s
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Factors Involved in Rapid Src
Activation
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Integrin activation
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Substrate stiffness
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Deep cytosol stimulation requires stress
fibers from focal adhesions
Prestress in actin is necessary for stress
transmission
F-actin integrity
What else…
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Methods: Microtubule, Actin
and Endosome Deformation
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1. Transfect cells with Src reporter and
mCherry-tubulin
2. Induce stress
3. Measured Src activation and deformation
of component in question
4. Repeat above with Src reporter and
mCherry-actin
5. Repeat above with mCherry-tubulin and
GFP-endo
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Results: Microtubules
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Results: Actin Deformation
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Results: Endosomal and
Microtubulal Co-deformation
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Conclusion
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Rapid Src activation too fast to be caused by
either diffusion or translocational-based
mechanisms. Thus, it is unique to
mechanotransduction.
Mechanism shown to be: Local stress at focal
adhesionF-actin bundles transmit stress to
deep cytoplasmMicrotubules and
endosomes deformSrc activation
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Questions?
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