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