Emergent structures in mixed lipid membranes

Emergent structures in mixed lipid membranes

Understanding
Structure formation in
Membranes
Lateral Fluidity
Out-of-plane dynamics
Chemical heterogeneity
Atul N. Parikh
[email protected]
Self-Assembly: Hydrophobic effect
MULTILAMELLAR
VESICLE
UNILAMELLAR
VESICLE
AMPHIPHILE
BILAYER
LIQUID-CRYSTALLINE
LAYERED STACKS
1
10
LOG (LENGTH SCALE), NM
100
1,000
(A )
D
W ATER
Supported Bilayer Lipid
Membranes
D
D
(B )
Figure 5 | Synthetic Model of the Cell Membrane. The cell membrane,
being a self-assembled fluid, can be reconstituted from its components such as shown.
By selecting appropriate lipids and proteins, a synthetic control of membrane structure
and function can be achieved.
Membrane Photolithography
QUARTZ TEMPLATE
CHROME LAYER
AQUEOUS PHASE (PBS)
HYDRATION LAYER
SILICA SUBSTRATE
100 um
UV (184-257 nm)
PBS
PBS
PBS
100 um
C. K. Yee, M. L. Amweg, A. N. Parikh, Adv. Mater. (2004)
C. K. Yee, M. L. Amweg, A. N. Parikh, JACS (2004)
Preserved fluidity of the unexposed bilayer
Fluorescence Recovery after Photobleaching (FRAP)
T = 0 min
T = 3 min
T = 5 min
Unexposed region
UV illuminated region
2750
2800
2850
2900
2950
Wavenumbers, cm-1
3000
3050
T = 9 min
Laterally Confined Photochemistry
(Wentworth et al, Science, 2002)
“Most membrane proteins do not enjoy the continuous
unrestricted lateral diffusion.... Instead, proteins diffuse in a
more complicated way that indicates considerable lateral
heterogeneity in membrane structure, at least on a nanometer
scale”
Jacobson, K., Sheets, E. D. & Simson, R. (1995) Science 268, 1441-1442
Multiphoton Photolithography
800 nm
100 fs
Patterning with Femto-sec Laser
Fluorescence images;
100X oil NA 1.45
97%POPC 3%NDB-PE
20μm
Before
Laser Exposure
Parameters
•λ = 800nm
•Energy per Pulse (6-12nJ per pulse
•Rep Rate = 540 KHz
•Focus Diameter = 0.5μm
After
(modified)
Multiphoton Modification of Lipids
“Erasing” Barriers
Repetition Rate Dependance
• 2.7kHz
• 1 laser pulse approx. every 10-4s
• Able to pattern
• 540kHz
• 1 laser pulse approx. every 10-6s (~200x)
• Able to pattern and with same power, “erase”
• Same power thresholds
Confined box
Re-exposed
perimeter
Portion is
removed
time
Box is now open
Probing Membrane Heterogeniety and Dynamics
using model bilayers
PBS
PBS
PBS
PBS
PBS
100 um
Before
Backfilling
T=0 min.
T=1 min.
T=5 min.
T=10 min.
T=20 min.
A Biophysical tool
for Understanding
Lipid heterogeneity
Designed reactive-diffusive
fronts
Lipid-lipid interdiffusion,
compositional manipulation
Phase dynamics and stability
Engineering arrested diffusion
Kinetically and chemically arrested
Mixing for functional patterning
HAEC
Gm1
HL-60
CTB
Bilayers
CTB
Engineering Lipid Rafts
Sphingomyelin
Cholesterol
Gm1
Phospholipid
PBS
PBS
PBS
PBS
PBS
A. R. Sapuri, J. T. Groves, A. N. Parikh, in preparation (2004)
TEMPLATED SELF-ASSEMBLY OF PHOSPHOLIPID
MEMBRANES
JACS 2005
a
b
d
c
e
1:1:1, DOPC:SM:Chol
3% NBD DHPE, 32%DOPC, 31% cholesterol, 32% sphingomyelin, 2% GM1
Ex 0.9sec
a
b
c
POPC GM1 +fitc CTB
TR POPC GM1 + FITC CTB
TR image
Fitc image
TR POPC GM1 + FITC CTB
Green Channel image
Corrected fret image
Figure 3
a
c
b
d
e
100μm
f
c
Buckling of PDMS Stamp
Quartz
Chrome
PDMS Stamp
Quartz Chrome
Photomask
UV/Ozone
PDMS
Quartz
Chrome
Buckling of the
PDMS Stamp
SiOx
Peel the PDMS
from the Mask
Photochemically buckled PDMS
substrates
80
60
1
µm
µm
0
40
-1
20
-2
0
0
20
40
µm
60
80
500um Pattern
1.0
80
0.0
60
-0.5
1
0
40
µm
µm
Height
0.5
-1.0µm
-1
20
0
20
40
60
µm
80
-2
100
0
0
20
40
µm
60
80
TR Labeled
Lipid
Incubation
2 minutes
Anisotropic recovery of Photobleached spot
Tm of DMPC is 23°C
Epifluorescense image: 1% Texas red DHPE, 99% DMPC
Comparison of FRAP on flat and Buckled Stamp
Tm of DMPC is 23°C
Epifluorescense image: 1% Texas red DHPE, 99% DMPC
COWORKERS
Dr. Chanel K. Yee, Materials
Dr. Annapoorna R. Sapuri-Butti, Chemistry
Dr. Sanhita Dixit, Physics
Michael Howland, Materials Science
Andreia Michelle Smith, Biophysics
Calvin Yang, Biomedical Engineering
Alan Szmodis, Biophysics
Amy Anderson, Biophysics
Ravi Butti, Materials
Adrian Brozell, Applied Science
Babak Sanii, Applied Science
Rita El-Khouri, Chemistry
Michelle Muha, Optical Science
Allen T. Lee, Optical Science
FUNDING
BES, Department of Energy
The NSF Center for Biophotonics, UC Davis
NIH
Lipid rafts in
Cell signaling
Dynamic assemblies rich in
sphingomyelin, cholesterol,
and saturated lipids
Micro-environment for
signaling molecules and their
complex formations
Signaling: bacterial
recognition, adhesion,
transport, localized oxidation,
Drug-binding GPCR proteins
Engineering Lipid Rafts
Sphingomyelin
Cholesterol
Gm1
Phospholipid
PBS
PBS
PBS
PBS
PBS
A. R. Sapuri, J. T. Groves, A. N. Parikh, in preparation (2004)
Detergent-Resistancy
Raft-raft coalescence
During apoptosis in
HL-60 Cells
A
(Inducer: tBH, label: alexa-CTB)
B
C
D
Boiling, Yee, Lincoln, Gilchrist, Yeh, Parikh, Morse, Submitted, 2005
Orthogonal membrane phases by selective extraction of bilayer
phases
a
b
c
d
e
Triton X100 experiment
After triton
Before triton
Backfilled NBD POPC