Cell adhesion to supported peptide-amphiphile bilayer membranes

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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
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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).
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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).
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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).
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Peptide biomaterials peptide-amphiphiles

• 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

Peptide amphiphiles
GRGDSP peptide - headgroup
Hydrophobic tail section

• 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

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Self-assembly Vesicle Fusion

• 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

Vesicle incorporating lipids and peptide
amphiphiles
Vesicle Solution on Surface
Vesicle Fusion
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Patterned Surfaces
Creating Multi-component patterned surfaces
Surfaces - Glass
Barriers - Proteins, e.g. BSA, deposited by
microcontact printing
Concentration Gradient - Microfluidic
parallel flow - Fabrication of Microchannels
Cell adhesion assays
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Results Patterned Bilayers
Grid-patterned Stamp
Patterned bilayer viewed by Fluorescence
Microscopy
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Results Cell Adhesion

• DOPC bilayer viewed by fluorescence and light
  microscopy

Cells spread to clean glass surfaces but not to
fluid lipid bilayers
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Current work

• Cell adhesion to bilayers containing
  peptide-amphiphiles
• Fabrication of microchannels for creating
  patterned surfaces

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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?

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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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

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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).
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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).
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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).
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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).
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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).
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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).
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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).
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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

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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

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Fmoc Solid-Phase Peptide Synthesis