preview

tentchoirΤεχνίτη Νοημοσύνη και Ρομποτική

15 Νοε 2013 (πριν από 3 χρόνια και 11 μήνες)

84 εμφανίσεις

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

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).

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).


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 Fibronectin

binding to cell
-
surface integrins

Giancotti, FG,
et al
.,
Science
, 285, 1028 (1999).

Peptide biomaterials:

peptide
-
amphiphiles

O
C
O
O
C
O
N
C
H
O
C
O
N
H
C
O
N
H
C
O
N
H
N
H
2
H
N
N
H
C
O
N
H
C
O
C
H
O
O
N
H
C
O
O
H
N
H
C
O
O
H
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


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
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).

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
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).

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
t
r
C
D
t
t
r
C






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