Effects of Fluorescent Probes on Lipid Membrane Physical Properties
Sherry Leung , Jonathan Brewer , Luis Bagatolli and Jenifer Thewalt
1
1
2
2
1,3
Department of Physics, Simon Fraser University, Burnaby BC
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
3
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby BC
Motivation: Trace Impurities in Lipid Membranes
Many biological molecules are present in cells in trace amounts, and yet they
play important functional roles. Examples include caveolin fragments, PIP2
and GM1 [2, 3, 5]. Other studies have shown that minor impurities can alter
lipid membrane phase diagrams [4, 6, 8, 9].
2
Two Photon Excitation Fluorescence Microscopy of
Giant Unilamellar Vesicles
2
H NMR Spectroscopy of Multilamellar Vesicles
Ld-lo phase separation at the equatorial plane observed using the fluorescent
probe Laurdan.
Ld
Lo
The Big Question
Do minor components present in trace amounts alter liquid disorderedliquid ordered (ld-lo) membrane phase behavior?
Membrane Composition
0
0.206
0.707 0.206
GP
0.571 0.419
GP
0.707
Microscopy: Counting Phase Separated Vesicles
Laurdan Emission Spectrum and General Polarization
50
40
30
20
10
0
18
20
22
24
26
Temperature (°C)
28
Microscopy: General Polarization
350 400 450 500 550 600
Wavelength (nm)
A: Gel DPPC at 35 C
◦
B: Liquid crystalline DPPC at 50 C
Figure from [7].
◦
30 35 40 45 50 55 60
Temperature (C)
7
6
5
4
4
8
15 20 25 30 35 40
0%
0.25%
7
8
15 20 25 30 35 40
0%
0.5%
7
6
20
22
24
5
5
4
4
15 20 25 30 35 40
Outstanding Questions
• Is
15 20 25 30 35 40
H NMR: Overall Membrane Fluidity
0.2% #1
26
0.2% #1
28
0.35%
0.5
0.4
0.3
0.2
20
22
0.6
24
26
28
18
20
22
24
26
0.2% #2
0.5
0.4
More
Ordered
0.3
0.2
20
22
24
26
28
Temperature (ºC)
28
Both fluorescence microscopy and 2H NMR show that trace amounts
of the fluorescent probe Laurdan does not affect membrane phase behaviour. Laurdan can be used to measure the phase behaviour of 35:35:30
DPPC:DOPC:Chol without modifying it. This is in contrast to several other
recently-studied probes. We hypothesize that Laurdan’s ability to partition
nearly equally among different lipid phases is responsible for this difference.
6
75kHz
X
homogeneous
vesicles
ld domains
0.3
18
6
|ω|I(ω)
ω=−75kHz
75kHz
X
it generally true that trace components that partition equally between
ld and lo phases do not alter phase behaviour?
• Which biological molecules present in trace amounts in membranes partition unequally between ld and lo phases?
References
[1] L. Bagatolli, Springer Ser. Fluoresc. 13, 3 (2013).
.
[2] A. Hammond, F. Heberle, T. Baumgart, D. Holowka, B. Baird, G. Feigenson, PNAS,
102, 6320 (2005).
2I(ω)
ω=−75kHz
0.4
18
0%
0.375%
7
5
0.5
0.6
Sample Averaged GP
B
GP
Emission Intensity
A
0.6
0.5
0.4
0.3
0.2
0.1
0.0
- 0.1
- 0.2
8
0%
0.125%
M1 =
0.2
18
I440 I490
2
Average Spectral Width (kHz)
Single Vesicle GP
When Laurdan intercalates into a more disordered part of the membrane,
its emission spectrum is red shifted, decreasing the general polarization
(GP) [1].
I440 − I490
GP =
I440 + I490
lo domains
8
Temperature (°C)
For phase separated vesicles, the GP of ld and lo regions are measured separately. For homogenous vesicles, the GP for the entire vesicle is measured.
The GP for multiple vesicles in a given sample are then averaged.
0.6
Summary
x10
60
3
% Laurdan
0.2% Sample #1
0.2% Sample #2
0.35%
x10
35
DPPC-D62
70
H NMR: Flexibility of Membrane Interior
It is currently accepted that nano-scale lateral compositional heterogeneity plays functional roles in cell membranes. Ordered and disordered lipid
phases can coexist at appropriately chosen compositions, temperatures and
pressures. Fluorescence is a popular family of techniques used to study membranes, however recent systematic studies show that fluorescent probe behaviour can be altered by membrane composition, probe concentration, and
the presence of other probes. Using deuterium nuclear magnetic resonance
2
spectroscopy ( H NMR), we found that trace amounts of the carbocyanine
probe DiIC12 are enough to alter phase coexistence behaviour of 35:35:30
DPPC-D62:DOPC:cholesterol membranes, while other probes like Laurdan,
Naphthopyrene, and another carbocyanine probe DiOC18, did not affect
the membrane appreciably. Laurdan is particularly well suited to the study
of phase separation in lipid membranes. It partitions equally well into ordered and disordered lipid phases, and displays a phase-dependent emission
spectral shift. Laurdan general polarization (GP) parameter, which characterizes said emission spectral shift, has been used to characterize membrane
fluidity. We examine the relationship between Laurdan GP and 2H NMR
order parameters.
3
30
35
Chol DOPC
% Phase Separated Vesicles
2
3
3
Methyl Quadrupolar
Splitting
x10
x10 (kHz)
ε% Laurdan
GP
Abstract
[3] M. Horton, J. Rädler, A. Gast, Colloid and Interface Sci., 304, 67 (2006).
[4] J. Juhasz, J. Davis, F. Sharom, Biochem., 430, 415 (2010).
95
[5] A. Liu and D. Fletcher, Biophys. J., 91, 11 (2006).
% Laurdan
0%
0.125%
0.25%
0.375%
0.5%
90
85
[6] G. Putzel and M. Schick, Biophys. J., 96, 4935 (2009).
[7] S. Sánchez, E. Gratton, Acc. Chem. Res., 38, 469 (2005).
[8] M. Skaug, M. Longo, R. Faller, J. Phys. Chem., 115, 8500 (2011).
[9] S. Veatch, S. Leung, R. Hancock, J. Thewalt, J. Phys. Chem., 111, 502 (2007).
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Download this poster from www.sfu.ca/jthewaltlab.
More Ordered
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15
Acknowledgments
20
25
30
35
Temperature (°C)
40
LAB and JB want to thank the Danish National Research Foundation (which supported
MEMPHYS-Center for Biomembrane Physics), and DaMBIC (Danish Molecular Biomedical Imaging Center) for the use of its instrumental facility. S.L. was supported by an
NSERC Canada Graduate Scholarship and a Michael Smith Foundation Foreign Study
Supplement, and Simon Fraser University Graduate Fellowships and Graduate (International) Research Travel Award. J.T. was supported by an NSERC Discovery Grant. We
thank Mike Kirkness and Bashe Bashe for their assistance with experiments and analysis.