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 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 Corresponding Picture of a Human Oxygen We have a characteristic shape A boundary (skin) Are made of characteristic set of elements Carbon 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… 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… 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… 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 k BT r ( ) * G ( ) 2 Particle motion is determined by Brownian motion and mechanical stiffness Cell r 2 ( ) ( ) G ( ) * 2 Particle motion is determined motor activity and mechanical stiffness Kinesin and dynein activity Observe molecular processes with microscopy Endogenous particles tracked with ~ 3 nm and 90 s 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 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 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 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.” 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 Now Begin More Formal Mechanotransduction: Molecular Level How can force effect molecules? Conformational Change Hoffman, Nature, 2011 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 Wang, Nature, 2005 1 min force 5 min force 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 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 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 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 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 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 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…) human disease (metastatic cell migration, atheroslcerotic development…) Parsons, Nat Rev Mol Cell Bio, 2010 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 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 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 Force Across Vinculin Determines FA Dynamics Leading Edge High Tension Across Vinculin Low Tension Across Vinculin Trailing Edge 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 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? Questions?