Mechanical Force Effects on Cellular Function

BioE506 – 3.29.2008

James Eddy

Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize Alexander Bershadsky, Michael Kozlov, and Benjamin Geiger

Review Article Outline I.

Background – mechanosensitive adhesions II. Focal adhesions (refresher) III. Focal complex-to-adhesion transitions IV. Non-focal adhesion mechanosensors V. Mechanisms of force-dependent transition VI. Physical models

Mechanosensitive Adhesions • Cells sense not only ligands & adhesion molecules, but mechanical cues: – mechanical forces (stresses) – deformations (strains) • “Just-in-time” mechanosensing – external perturbations unpredictable in extent and timing – strength of adhesion should adjust itself dynamically to amount of stress/strain applied to site

Focal Adhesions • FAs associated with actin filaments in the cell • Interact with extracellular matrix via integrins • Evolve from focal complexes (FXs)

• Force-dependent FX-to-FA Transition The evidence: – Inhibition of myosin-II-driven contractility leads to accumulation of FXs and disappearance of FAs – Application of external force to FAs stimulates growth in direction of force – even with suppressed myosin II – Size of FAs and forces applied to them usually proportional – On soft matrix (strong forces not generated), large FAs not formed

Non-FA Mechanosensing Adhesions • Integrin-mediated – fibrillar adhesions: • • evolve from FAs in force-dependent manner do not assemble when force is relaxed – podosomes • lifespan, not shape, depends on substrate flexibility • Cadherin-mediated – cell-cell adherens junctions (AJs) – Platelet endothelial cell adhesion molecule (PECAM)

Molecular Mechanisms • Uncertain which molecular interactions (protein protein) are regulated by force • Possible signaling mechanisms: – Modulation of phosphorylation-dependent protein-protein interactions – Recruitment/translocation of adaptor proteins – Regulation of GTPase pathways

• Physical Modeling In absence of detailed signaling models, scientists have used physical models to formulate “ground rules” for mechanosensitivity • Three specific models described: – Stress sensing – – Strain sensing Thermodynamic • Varying assumptions in three models: – Presence of a molecular “switch” – Location of mechanosensors – Protein dynamics

Stress-sensing Model • Protein switch: dragging force causes conformational change of mechanosensitive protein • Possible mechanism for maturation of FXs to FAs via ECM elasticity

Strain-sensing Model • Protein switch: compression of mechanosenosensitive protein layer leads to growth (front) and disassembly (rear) of adhesion plaque • “Crawling” or “treadmilling” motion • Few relative protein changes

Thermodynamic Model • Elastic stress within plaque decreases chemical potential, enhancing self-assembly by addition of new plaque molecules • Internal “treadmilling-like” motion of proteins which can progress in different directions • Abundant relative protein changes

Review Article Summary • • • • Cells can sense mechanical stimuli, not just ligands or other molecules Focal adhesions are one key example of mechanosensitive cell adhesion Much is still unknown about protein interaction mechanisms in mechanosensitivity Three models (stress-sensing, strain-sensing, thermodynamic), postulate physical “rules” for mechanosensitivity

Forced Unfolding of Proteins Within Cells Colin P. Johnson, Hsin-Yao Tang, Christine Carag, David w. Speicher, Dennis E. Discher

Research Article Outline I.

Introduction II. Spectrin proteins (red blood cells) III. Cysteine labeling technique IV. Shear vs. Static labeling results (spectrin) V. Labeling dynamics VI. Differential labeling in mesenchemal stem cells

Force-induced Protein Unfolding • Reversible domain unfolding has been shown to occur in adhesion proteins in response to external forces • Direct cell-level evidence is lacking, and a new “shotgun” approach for cysteine residue labeling is presented

Red Blood Cell – Spectrin Proteins • Spectrin proteins have been proven central to red blood cell deformability under stresses of blood flow • α and β chains crosslink with F-actin, and helical bundle domains unfold at low forces

Cysteine Labeling • Cysteine (Cys) residues are moderately hydrophobic, and often hidden in tertiary or quaternary protein structure • Protein unfolding allows labeling of newly exposed Cys residues by Cys-reactive phlorophore IAEDANS • Label phlorophores interact with exposed SH groups

Cysteine Labeling • Shotgun in situ labeling: – Cells reversibly lysed, then resealed after entrapment of phlorophore IAEDANS – Dye-loaded cells either held static (with varying temperature), or sheared over physiological range of stresses – Cells relysed and imaged – Cells denatured, non-labeled Cys were alkylated with iodoacetamide (IAM) – Membranes separated by 1D SDS-PAGE

Shear vs. Static Cys Labeling • α and β chains show significant increase in Cys residue labeling when exposed to shear stresses • Other membrane proteins remain relatively unchanged

Shear vs. Static Cys Labeling • Liquid chromatography-coupled tandem mass spectrometry (LC MS/MS) was used to identify and quanitfy Cys-modified sites in spectrin bands (after excision, trypsinization)

Heat-induced Unfolding • Recombinant spectrin proteins were analyzed under varying temperatures

Labeling Dynamics • Sequential 2-dye labeling improves differential labeling

Mesenchemal Stem Cells • MSCs are contractile and strain underlying matrix in differentiation (stresses ~1000 times higher than fluid on RBCs) • Inhibition of non-muscle myosin II (NMM II) with the drug blebbistatin relaxes and softens cells, prevents differentiation

MSC Cys Labeling

MSC Cys Labeling • Much higher Cys labeling in tensed vs. relaxed NMM II (analogous to shear vs. static) • Blebbistatin induced depolymerization of vimentin and actin increases Cys labeling

Differentially Labeled NMMII Cys Sites • Homology model shows Cys 90 , identified as differentially labeled, to be buried between head and rod domains

Cys-labeling of Monomers/Polymers • It was shown that Cys labeling is enhanced for monomeric proteins (which agrees with depolymerization results)

Potential Cys-labeling Applications • Time-integrated Cys labeling techniques might be combined with other real-time imaging methods (e.g. FRET) • Could be used to correlate unfolding with post-translational modifications (i.e. phosphorylation)

Summary & Criticisms • Certain membrains do unfold under physical forces • Cys labeling is an effective method for identifying and quantifying newly exposed protein regions following unfolding