Cell adhesion to supported peptide-amphiphile bilayer membranes Badriprasad Ananthanarayanan Advised by Matthew Tirrell Ph...
Introduction <ul><li>Biomaterials </li></ul><ul><ul><li>Surface functionalization for increased compatibility and safety <...
Biomimetics <ul><li>Engineering biological recognition to create ‘biomimetic’ materials </li></ul><ul><li>Extra-Cellular M...
RGD and Integrins <ul><li>Fibronectin is one of the adhesion-promoting proteins in the ECM </li></ul><ul><li>Fibronectin b...
Peptide biomaterials: peptide-amphiphiles Hydrophobic ‘tail’ section Peptide amphiphiles <ul><li>Peptide headgroups coval...
Self-assembly: Vesicle Fusion Vesicle Fusion Vesicle Solution on Surface Vesicle incorporating lipids and peptide amphiphi...
Patterned Surfaces Surfaces: - Glass Barriers: - Proteins, e.g. BSA, deposited by microcontact printing Concentration Grad...
Results: Patterned Bilayers Grid-patterned Stamp Patterned bilayer viewed by Fluorescence Microscopy
<ul><li>DOPC bilayer viewed by fluorescence and light microscopy </li></ul>Results: Cell Adhesion Cells spread to clean gl...
Current work <ul><li>Cell adhesion to bilayers containing peptide-amphiphiles </li></ul><ul><li>Fabrication of microchanne...
Effect of Membrane Fluidity on Cell Adhesion <ul><li>SLBs used in our research as a platform for incorporating adhesion-pr...
Membrane fluidity in nature <ul><li>Fluid Mosaic model of membranes – proteins and lipids have varying degrees of lateral ...
Example: Immune Recognition <ul><li>T-cell activation is a critical step in the immune response </li></ul><ul><li>T-cell a...
Influence of Ligand Mobility <ul><li>T-cell receptor CD2 and its counter-receptor CD58 (LFA-3) – one of the receptor-ligan...
Cell adhesion: RGD and integrins <ul><li>Integrins association with ECM is essential for cell adhesion and motility </li><...
Effect of RGD clustering <ul><li>The effect of RGD surface density is well known </li></ul><ul><ul><li>Average ligand spac...
Effect of RGD clustering <ul><li>There is a definite effect of nanoscale clustering of ligands on cell adhesion </li></ul>...
Simulation of RGD clustering <ul><li>Single-state model – clustering of ligands does not change binding affinity K D </li...
Effect of bilayer fluidity <ul><li>Spatial organization of ligand has a great effect on cell adhesion, hence fluidity of S...
SLB – controlling fluidity <ul><li>Polymerizable Lipid tails </li></ul><ul><ul><li>Diacetylenic moieties in lipid tails – ...
SLB – controlling fluidity <ul><li>Quenching mixed-lipid bilayers below the melting temperature </li></ul><ul><ul><li>e.g....
Characterizing Fluidity – FRAP <ul><li>Fluorescence Recovery After Photobleaching </li></ul><ul><li>Fluorescent molecules ...
FRAP – analysis <ul><li>Diffusion equation for one species </li></ul><ul><li>Solution: Gaussian beam intensity profile, ci...
FRAP – instrument setup <ul><li>Light source: High-power lamp or laser </li></ul><ul><li>Electromechanical shutter system ...
Cell adhesion assays <ul><li>Determining adhesion strength </li></ul><ul><li>Centrifugal detachment assay </li></ul><ul><u...
Cell adhesion assays <ul><li>Detect extent of cytoskeletal organization and focal adhesion assembly </li></ul><ul><li>Stai...
Conclusions <ul><li>Constructing supported bilayer membranes incorporating peptide-amphiphiles for cell adhesion </li></ul...
Phase separation <ul><li>Lateral phase separation may be important in the SLB </li></ul><ul><li>Solid-phase lipid domains...
Fmoc Solid-Phase Peptide Synthesis
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  • 1. Cell adhesion to supported peptide-amphiphile bilayer membranes Badriprasad Ananthanarayanan Advised by Matthew Tirrell PhD Candidacy exam, August 2004 Faculty Committee: Matthew Tirrell Jacob Israelachvili Samir Mitragotri Luc Jaeger
  • 2. Introduction <ul><li>Biomaterials </li></ul><ul><ul><li>Surface functionalization for increased compatibility and safety </li></ul></ul><ul><ul><li>Examples </li></ul></ul><ul><ul><li>Implant materials, e.g. Vascular grafts </li></ul></ul><ul><ul><li>Seeding with endothelial cells improves </li></ul></ul><ul><ul><li>graft performance </li></ul></ul><ul><ul><li>Tissue engineering scaffolds </li></ul></ul><ul><ul><li>Cells require many signals from matrix to enable </li></ul></ul><ul><ul><li>proliferation and tissue regrowth </li></ul></ul>Tirrell, M et al. , Surface Science , 500 , 61 (2000).
  • 3. Biomimetics <ul><li>Engineering biological recognition to create ‘biomimetic’ materials </li></ul><ul><li>Extra-Cellular Matrix </li></ul><ul><li>Proteins in the ECM e.g. fibronectin and others </li></ul><ul><li>provide a structural framework and biochemical </li></ul><ul><li>signals that control cellular function, e.g. adhesion, </li></ul><ul><li>growth, differentiation, etc. </li></ul><ul><li>Creating biomaterials which reproduce these interactions </li></ul><ul><li>may allow us to direct cell adhesion </li></ul>Tirrell, M et al. , Surface Science , 500 , 61 (2000).
  • 4. RGD and Integrins <ul><li>Fibronectin is one of the adhesion-promoting proteins in the ECM </li></ul><ul><li>Fibronectin binds to cell-surface receptors known as integrins , trans-membrane proteins which regulate a number of cellular processes </li></ul><ul><li>The binding site for many integrins in fibronectin is the loop containing the peptide sequence Arg-Gly-Asp (RGD) </li></ul>RGD sites on Fibronectin binding to cell-surface integrins Giancotti, FG, et al ., Science , 285, 1028 (1999).
  • 5. Peptide biomaterials: peptide-amphiphiles Hydrophobic ‘tail’ section Peptide amphiphiles <ul><li>Peptide headgroups covalently linked to a hydrophobic ‘tail’ segment </li></ul><ul><li>Hydrophobic-force driven self-assembly into micelles, vesicles, bilayers, etc. allows us to easily deposit functional molecules on surfaces using self-assembly </li></ul><ul><li>Short peptides incorporating the RGD sequence can bind integrins and promote cell adhesion, similar to fibronectin </li></ul><ul><li>Using peptides may offer advantages over proteins in terms of convenience, selectivity, and presentation on surfaces </li></ul>GRGDSP peptide - headgroup
  • 6. Self-assembly: Vesicle Fusion Vesicle Fusion Vesicle Solution on Surface Vesicle incorporating lipids and peptide amphiphiles <ul><li>Vesicles are formed from a solution of amphiphiles </li></ul><ul><li>When exposed to a hydrophilic surface, vesicles rupture and form bilayer fragments which fuse to form a continuous bilayer on the surface </li></ul><ul><li>Clean hydrophobic surfaces are essential for fusion, smaller vesicles are more fusogenic </li></ul>Hydrophilic Substrate
  • 7. Patterned Surfaces Surfaces: - Glass Barriers: - Proteins, e.g. BSA, deposited by microcontact printing Concentration Gradient: - Microfluidic parallel flow - Fabrication of Microchannels Cell adhesion assays Creating Multi-component patterned surfaces Lipid Peptide amphiphile
  • 8. Results: Patterned Bilayers Grid-patterned Stamp Patterned bilayer viewed by Fluorescence Microscopy
  • 9. <ul><li>DOPC bilayer viewed by fluorescence and light microscopy </li></ul>Results: Cell Adhesion Cells spread to clean glass surfaces but not to fluid lipid bilayers Control glass surfaces for comparison :
  • 10. Current work <ul><li>Cell adhesion to bilayers containing peptide-amphiphiles </li></ul><ul><li>Fabrication of microchannels for creating patterned surfaces </li></ul>
  • 11. Effect of Membrane Fluidity on Cell Adhesion <ul><li>SLBs used in our research as a platform for incorporating adhesion-promoting ligands </li></ul><ul><ul><li>Ease of fabrication by vesicle fusion </li></ul></ul><ul><ul><li>Inert background: cells show no adhesion to fluid lipid bilayers </li></ul></ul><ul><ul><li>Retains lateral mobility of membrane components and hence a better mimic of cell membrane </li></ul></ul><ul><li>Fluidity of SLBs has been used for various purposes </li></ul><ul><ul><li>Creating micropatterned surfaces </li></ul></ul><ul><ul><li>Biosensors, etc. </li></ul></ul><ul><ul><li>Does the fluidity have an effect on cell adhesion? </li></ul></ul>
  • 12. Membrane fluidity in nature <ul><li>Fluid Mosaic model of membranes – proteins and lipids have varying degrees of lateral fluidity </li></ul><ul><li>Lateral mobility of membrane proteins is an essential step in many signal transduction pathways, e.g. action of soluble hormones, immune recognition, growth, etc. </li></ul>Jacobson, K et al. , Science 268, 1441 (1995).
  • 13. Example: Immune Recognition <ul><li>T-cell activation is a critical step in the immune response </li></ul><ul><li>T-cell activation requires sustained engagement of T-cell receptors by ligands through the ‘immunological synapse’ </li></ul><ul><li>Formation of this structure involves many receptor-ligand pairs and their transport within the membrane </li></ul>Groves, JT et al. , J. Immunol. Meth. 278, 19 (2003).
  • 14. Influence of Ligand Mobility <ul><li>T-cell receptor CD2 and its counter-receptor CD58 (LFA-3) – one of the receptor-ligand pairs involved in T-cell signalling </li></ul><ul><li>CD58 found in two forms: lipid-anchored (GPI) and transmembrane (TM) </li></ul><ul><li>lipid-anchored form was mobile, TM form immobile </li></ul><ul><li>Adhesion of T-cells to GPI-anchored form at lower densities, and adhesion strength also higher </li></ul>Chan, P-Y et al. , J. Cell. Bio. 115, 245 (1991).
  • 15. Cell adhesion: RGD and integrins <ul><li>Integrins association with ECM is essential for cell adhesion and motility </li></ul><ul><li>Integrins cluster as they bind, enabling assembly of their cytoplasmic domains which initiates actin stress fiber formation </li></ul><ul><li>This results in more integrin clustering, binding and finally, formation of focal contacts essential for stable adhesion </li></ul>Ruoslahti, E et al. , Science 238, 491 (1987); Giancotti FG et al. , Science 285, 1028 (1999).
  • 16. Effect of RGD clustering <ul><li>The effect of RGD surface density is well known </li></ul><ul><ul><li>Average ligand spacing of 440 nm for spreading, 140 nm for focal contacts </li></ul></ul><ul><li>Some evidence that clustering of ligands facilitates cell adhesion </li></ul><ul><ul><li>(RGD)n-BSA conjugates show equivalent adhesion at much lower RGD densities for higher values of n </li></ul></ul><ul><ul><li>Synthetic polymer-linked RGD clusters show more efficient adhesion and well-formed stress fibers for nine-member clusters </li></ul></ul>Danilov YN et al., Exp. Cell Res. 182, 186 (1989).
  • 17. Effect of RGD clustering <ul><li>There is a definite effect of nanoscale clustering of ligands on cell adhesion </li></ul>Maheshwari G et al., J. Cell Sci. 113, 1677 (2000).
  • 18. Simulation of RGD clustering <ul><li>Single-state model – clustering of ligands does not change binding affinity K D </li></ul><ul><ul><li>No effect observed on ligand clustering other than receptor clustering </li></ul></ul><ul><li>Two-state model – ligand clustering causes increase in K D – represents activation of receptor in vivo </li></ul><ul><ul><li>Significantly higher number of receptors bound, especially at low average ligand density </li></ul></ul><ul><ul><li>This translates into stronger adhesion and better assembly of focal contacts </li></ul></ul>Irvine, DJ et al., Biophys. J. 82, 120 (2002).
  • 19. Effect of bilayer fluidity <ul><li>Spatial organization of ligand has a great effect on cell adhesion, hence fluidity of SLB may have an effect </li></ul><ul><li>Experimental plan </li></ul><ul><ul><li>Controlling fluidity in SLBs </li></ul></ul><ul><ul><li>Characterizing fluidity – FRAP </li></ul></ul><ul><ul><li>Cell adhesion assays </li></ul></ul><ul><ul><li>SLB microstructure – formation of domains </li></ul></ul>
  • 20. SLB – controlling fluidity <ul><li>Polymerizable Lipid tails </li></ul><ul><ul><li>Diacetylenic moieties in lipid tails – can be polymerized by UV irradiation </li></ul></ul><ul><ul><li>Polymerizable tails can be conjugated to RGD, or lipids with polymerizable tails can be used as a background </li></ul></ul><ul><ul><li>Control fluidity by varying the degree of polymerization as well as the concentration of polymerizable molecules </li></ul></ul>Tu, RS, PhD thesis, UCSB (2004).
  • 21. SLB – controlling fluidity <ul><li>Quenching mixed-lipid bilayers below the melting temperature </li></ul><ul><ul><li>e.g. mixed DLPC/DSPC vesicles quenched from 70 0 C to room temperature </li></ul></ul><ul><ul><li>Results in formation of small lipid domains </li></ul></ul><ul><ul><li>These domains act as obstacles to lateral diffusion in the bilayer </li></ul></ul><ul><ul><li>When solid-phase area fraction is very high, diffusion of fluid-phase molecules goes to zero </li></ul></ul>Ratto TV et al ., Biophys J. 83, 3380 (2002).
  • 22. Characterizing Fluidity – FRAP <ul><li>Fluorescence Recovery After Photobleaching </li></ul><ul><li>Fluorescent molecules bleached by high-intensity light source or laser pulse </li></ul><ul><li>The same light source, highly attenuated, is used to monitor recovery of fluorescence due to diffusion of fluorescent molecules into the bleached area </li></ul><ul><li>Spot bleaching or Pattern Bleaching </li></ul><ul><li>Curve fitting gives diffusion constant and mobile fraction </li></ul>Groves, JT et al. , Langmuir 17, 5129 (2001).
  • 23. FRAP – analysis <ul><li>Diffusion equation for one species </li></ul><ul><li>Solution: Gaussian beam intensity profile, circular spot </li></ul><ul><li>Curve fitting gives diffusion constant </li></ul>Axelrod, D et al. , Biophys J . 16, 1055 (1976); Ratto TV et al ., Biophys J. 83, 3380 (2002).
  • 24. FRAP – instrument setup <ul><li>Light source: High-power lamp or laser </li></ul><ul><li>Electromechanical shutter system used to switch between high-intensity beam and fluorescence observation light </li></ul><ul><li>PMT vs. Camera – camera allows spatial resolution of intensity, and hence we can monitor background fluorescence recovery, other transport processes </li></ul><ul><li>Data analysis by image-analysis software </li></ul>Meyvis, TLK, et al. , Pharm. Res. 16, 1153 (1999).
  • 25. Cell adhesion assays <ul><li>Determining adhesion strength </li></ul><ul><li>Centrifugal detachment assay </li></ul><ul><ul><li>Sample plate spun in centrifuge, adherent cells counted before and after </li></ul></ul><ul><ul><li>Low detachment forces applied </li></ul></ul><ul><li>Hydrodynamic flow </li></ul><ul><ul><li>Shear stress applied due to flow </li></ul></ul><ul><ul><li>Many configurations possible </li></ul></ul><ul><ul><li>Detachment force may depend on cell morphology </li></ul></ul>Garcia, AJ et al. , Cell Biochem. Biophys. 39, 61 (2003).
  • 26. Cell adhesion assays <ul><li>Detect extent of cytoskeletal organization and focal adhesion assembly </li></ul><ul><li>Staining of actin filaments to visualize stress fiber formation </li></ul><ul><li>Population of cells that show well-formed stress fibers can be visually determined </li></ul>Maheshwari, G et al., J. Cell. Sci. 113, 1677 (2000).
  • 27. Conclusions <ul><li>Constructing supported bilayer membranes incorporating peptide-amphiphiles for cell adhesion </li></ul><ul><li>Creating micropatterned surfaces for displaying spatially varied ligand concentrations </li></ul><ul><li>Effect of bilayer fluidity on cell adhesion strength and focal adhesion assembly </li></ul><ul><li>Design of efficient biomimetic surfaces for analytical or biomedical applications </li></ul>
  • 28. Phase separation <ul><li>Lateral phase separation may be important in the SLB </li></ul><ul><li>Solid-phase lipid domains may impart structural rigidity to the membrane, and/or anchoring sites for focal adhesions </li></ul><ul><li>Investigate by fluorescence microscopy, AFM </li></ul>
  • 29. Fmoc Solid-Phase Peptide Synthesis

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