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Cell adhesion to supported peptide-amphiphile bilayer
membranesBadriprasad Ananthanarayanan
Advised by
Matthew Tirrell
PhD Candidacy exam, August 2004
Faculty Committee:
Matthew Tirrell
Jacob Israelachvili
Samir Mitragotri
Luc Jaeger
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 scaffoldsCells require many signals from matrix to enable proliferation and tissue regrowth
Tirrell, M et al., Surface Science, 500, 61 (2000).
Biomimetics
Engineering biological recognition to create ‘biomimetic’ materials
Extra-Cellular Matrix
Proteins in the ECM e.g. fibronectin and othersprovide a structural framework and biochemicalsignals that control cellular function, e.g. adhesion,growth, differentiation, etc.
Creating biomaterials which reproduce these interactionsmay allow us to direct cell adhesion
Tirrell, M et al., Surface Science, 500, 61 (2000).
• 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 and Integrins
RGD sites on Fibronectinbinding to cell-surface integrins
Giancotti, FG, et al., Science, 285, 1028 (1999).
Peptide biomaterials: peptide-amphiphiles
O CO
O CO
N C
H
O
C
O
N
H
C
O
N
H
C
O
NH
NH2HN
N
H
C
O
N
H
C
O
C
HOO
N
H
C
O
OH
N
H
C
O
OH
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
Self-assembly: Vesicle Fusion
Vesicle Fusion
Vesicle Solution on Surface
Hydrophilic Substrate
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
Patterned Surfaces
Surfaces: - Glass Barriers: - Proteins, e.g. BSA,
deposited by microcontact printing
Concentration Gradient: - Microfluidic parallel flow - Fabrication of Microchannels
Lipid
Peptide amphiphile
Cell adhesion assays
Creating Multi-component patterned surfaces
Results: Patterned Bilayers
Grid-patterned Stamp
Patterned bilayer viewed by Fluorescence
Microscopy
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:
Current work
Cell adhesion to bilayers containing peptide-amphiphiles
Fabrication of microchannels for creating patterned surfaces
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?
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).
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).
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).
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).
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).
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).
Simulation of RGD clustering
Single-state model – clustering of ligands does not change binding affinity KD
No effect observed on ligand clustering other than receptor clustering
Two-state model – ligand clustering causes increase in KD – 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).
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
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).
SLB – controlling fluidity
Quenching mixed-lipid bilayers below the melting temperature e.g. mixed DLPC/DSPC vesicles quenched from
700C 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).
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).
FRAP – analysis
Diffusion equation for one species
Solution: Gaussian beam intensity profile, circular spot
Curve fitting gives diffusion constant
),(),( 2 trCD
t
trC
Axelrod, D et al., Biophys J. 16, 1055 (1976); Ratto TV et al., Biophys J. 83, 3380 (2002).
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).
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).
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).
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