Cell and Model Membrane Interactions

286 Pages·1991·11.169 MB·English

by  C. Tilcock, S. Eastman, D. Fisher (auth.), Shinpei Ohki (eds.)

Cell and Model
Membrane Interactions
Cell and Model
Membrane Interactions
Edited by
o
Shinpei hki
State University of New York at Buffalo
Buffalo, New York
SPRINGER SUIENUE+BUSINESS MEDIA, LLU
Llbrary of Congress Cataloglng-ln-Publlcatlon Data
Cell and model membrane inte~actions I edited by Shinpei Ohki.
p. cm.
"Proceedings of a symposlum on Cell and Model Membrane
Interactions, held Apri 1 22-27, 1990, in Boston, Massachusetts"
-T.p. versa.
Includes bibliographical references and index.
ISBN 978-1-4613-6720-8 ISBN 978-1-4615-3854-7 (eBook)
DOI 10.1007/978-1-4615-3854-7
i. Membrane fusion--Congresses. 1. Ohki, Shinpei.
OH60 1 . C33 1992
574.87" 5--dc20 91-43343
CIP
Proceedings of a symposium on Cell and Model Membrane Interactions,
held April 22-27, 1990, in Boston, Massachusetts
ISBN 978-1-4613-6720-8
© 1991 Springer Science+Business Media New York
Originally published by Plenum Press, New York in 1991
Softcover reprint of the hardcover 1 st edition 1991
AII rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
Preface
Membrane interaction is a large research area involving various disciplines.
A symposium entitled "Cell and Model Membrane Interactions" which took place in
Boston, MA during the 155th American Chemical Society Meeting, April 25, 1990,
focused on membrane adhesion and fusion. The topics were explored in studies
involving lipids, virus envelopes and cell membranes. Especially discussed, were
the roles of polymers, lipids, and proteins on these membrane interactions.
Fusion of membrane is an important molecular event which plays a pivotal
role in many dynamic cellular processes, such as exocytosis, endocytosis, membrane
genesis, viral infection processes, etc. The process includes adhesion of the mem
branes, fusion, and finally reorganization of the components of the two membranes.
The basic notion shared during the symposium was that membrane hydro
phobicity, especially local membrane hydrophobicity is one of the important factors
contributing to membrane fusion. Most of the papers are collected here and they
are arranged approximately in the same order as they were presented at the sympo
sium. These papers are the most up-to-date and representative work at the forefront
in each membrane interaction field.
I sincerely hope the reader will gain further understanding on membrane
interactions especially, membrane and vesicle fusion phenomena through this
symposium proceedings volume.
Shinpei Ohki
June 1991
v
Contents
Lipid Membrane Organization and Molecular Partitions
Detennination of Lipid Asymmetry and Exchange in Model
Membrane Systems .......................................... 1
C. Tilcock, S. Eastman, and D. Fisher
Partitioning of Gramicidin A' Between Coexisting Phases
within Phospholipid Bilayers .................................... 15
A.R.G. Dibble, M.D. Yeager, and G.W. Feigenson
Role of Macromolecules on Membrane Interaction
Membrane Contact Induced Between Erythrocytes by
Polycations, Lectins and Dextran ................................. 25
W.T. Coakley, H. Darmani, and A.J. Baker
Pegylation of Membrane Surfaces .................................... 47
D. Fisher, C. Delgado, J. Morrison,
G. Yeung, and C. Tilcock
Influence of Polar Polymers on the Aggregation
and Fusion of Membranes ..................................... 63
K. Arnold, M. Krumbiegel, O. Zschomig,
D. Barthel, and S. Ohki
Role of Lipids and Proteins on Membrane Adhesion and Fusion
Control of Fusion of Biological Membranes by Phospholipid
Asymmetry ................................................ 89
A. Herrmann, A. Zachowski, P.F. Devaux,
and R. Blumenthal
Annexin-Phospholipid Interactions in Membrane Fusion .................... 115
P. Meers, K. Hong, and D. Papaphadjopoulos
vii
Biological Consequences of Alterations in the Physical
Properties of Membranes ..................................... 135
R.M. Epand
Evidence for Multiple Steps in Enveloped Virus Binding ................... 149
A.M. Haywood
Inhibition of Sendai Virus Fusion and Phospholipid Vesicle
Fusion: Implications for the Pathway of Membrane Fusion . . . . . . . . . . . . .. 163
P.L. Yeagle, D.R. Kelsey, T.D. Flanagan, and
J. Young
Fusion of Influenza, Sendai and Simian Immunodeficiency
Viruses with Cell Membranes and Liposomes . . . . . . . . . . . . . . . . . . . . . . . 179
N. Diizgiine~, M.C. Pedroso de Lima, C.E. Larsen,
L. Stamatatos, D. Flasher, D.R. Alford,
D.S. Friend, and S. Nir
Physical Basis Underlying Membrane Adhesion and Fusion
Red Blood Cell Interaction with a Glass Surface 199
J.K. Angarski, K.D. Tachev, LB. Ivanov,
P.A. Kralchevsky, and E.F. Leonard
On the Mechanism of Membrane Fusion: Use of Synthetic
Surfactant Vesicles as a Novel Model System ....................... 215
T.A.A. Fonteijn, J.B.F.N. Engberts,
and D. Hoekstra
Kinetics of Intermembrane Interactions Leading to Fusion .................. 229
D.S. Dimitrov and R. Blumenthal
Short-Range Repulsive Interactions Between the Surfaces of
Lipid Membranes .......................................... 249
TJ. McIntosh, A.D. Magid, and S.A. Simon
Physico-Chemical Factors Underlying Membrane Adhesion
and Fusion ............................................... 267
S.Ohki
Index ...................................................... 285
viII
DETERMINATION OF LIPID ASYMMETRY AND EXCHANGE IN MODEL MEMBRANE SYSTEMS
C. Tilcock1, S. Eastman2 and D. F isher3
1 Department of Radiology, University of British Columbia
Vancouver, BC, Canada V6T 2B5
ZDepartment of Biochemistry, University of British Columbia
Vancouver, BC, Canada V6T lW5
3 Department of Biochemistry and Molecular Cell Pathology Laboratory
Royal Free Hospital School of Medicine, University of London
London NW3 2PF, UK
INTRODUCTION
Several biological membrane systems, including the erythrocyte and
platelet plasma membranesl-3, inner mitochondial membrane4 and endoplasmic
reticulum5 are known to exhibit asymmetry with respect to lipid
distribution across the lipid bilayer. For example, it is well established
that in the erythrocyte plasma membrane, the majority of the phosphatidyl
choline and sphingomyelin are present in the outer leaflet of the
bilayer while the aminolipids phosphatidylethanolamine and phosphat idyl
serine are located primarily on the inner leaflet. Despite studies which
implicate a role for specific lipid-transport proteins6-B in the
transbilayer movement of the aminolipids phosphatidylserine (PS) and
phosphatidylethanolamine (PE), the mechanisms responsible for the
generation and maintenance of lipid asymmetry in biological membranes
remain ill defined. The functional significance of such lipid asymmetry is
also not clear, although there is evidence that loss of phosphat idyl
serine asymmetry is correlated with increased macrophage uptake of
erythrocytes9 and also platelet activation10.
Studies in model membrane systems indicate that intrabilayer lipid
transport rates in response to putative modulators of lipid asymmetry such
as intrinsic and extrinsic proteinsll•12, phase separation phenomena 13 and
oxidative processes14 are often less than that observed for biological
systems15•16. Given the observation that fatty acids act as proton
ionophores17, it is of interest that fatty acids have been shown to
redistribute rapidly across a model membrane system in response to an
applied pH gradient1B. This observation has been recently extended to the
negatively-charged diacylphosPQolipids phosphatidylglycerol (PG)19 and
phosphatidic acid (PA)20, which redistribute across a lipid bilayer in
response to an applied pH gradient to accumulate on the high pH side of
the bilayer.
In this article a novel general procedure for the quantitative
determination of lipid asymmetry is described, based upon partition in
aqueous two-phase polymer systems. Partitioning in dextran-polyethylene
glycol two-phase systems is an established method for the fractionation of
cells and organelles on the basis of subtle differences in the interaction
Cell and Model Membrane Interactions
Edited by S. Ohki. Plenum Press. New York. 1991
of surfaces with the polymer systems21• Addition of anions such as alkali
phosphates or sulfates to aqueous two-phase polymer systems results in the
establishment of a liquid-junction (Donnan) potential between the two
phases (top phase positive) due to differential ion adsorption to each
phase22• This electrostatic potential difference may be utilized to
measure the charge on a particulate such as a lipid vesicle23-25• The
utility of the phase partition method is demonstrated using examples of
pH-gradient induced asymmetry in lipid vesicles containing stearylamine
(SA), PA, PG and cardiolipin (CL). The effects of temperature and lipid
composition upon both the rate of formation and extent of asymmetry in
model systems is described. Lastly, an example is given of one model
system in which the interaction between two membrane surfaces is modulated
by the lipid asymmetry within the individual membranes.
MATERIALS AND METHODS
Preparation of Lipid Vesicles
Lipids were combined in the appropriate molar ratios from stock
solutions in chloroform, solvent removed initially under nitrogen then by
storage under reduced pressure « 0.1 rom Hg) for at least one hour. For
partition measurements, the lipids were spiked with 1-2 ~Ci of tritiated
dipalmitoyl phosphatidylcholine 3H-DPPC prior to removal of solvent.
Multilamellar vesicles were prepared by dispersing 25 ~oles of lipid in
2 ml of either 10 roM sodium citrate pH 4, 10 roM sodium phosphate pH 7 or
10 roM sodium phosphate pH 8.5 by extensive vortexing at room temperature
then freeze-thawed five times from liquid nitrogen. Lipid vesicles of 100
nm average diameter were prepared by extrusion through polycarbonate
filters under nitrogen pressure as previously described26• A pH gradient
was established across the vesicle bilayers by column chromatography on
Sephadex G50M using the appropriate external buffer as eluant.
Partition Measurements
A phase system of 5% (w/w) dextran T-500 (Pharmacia) and 5% (w/w)
poly(ethylene glycol), PEG 8000 (BDH), was prepared in 10 roM sodium
phosphate at both pH 7 and pH 8.5. The phases were mixed and allowed to
equilibrate at 25°C overnight. The PEG-rich upper phase and dextran-rich
lower phase were separated and stored at -20°C. For partition
measurements, typically 30 ~l of vesicle preparation (0.3-0.4 ~oles
lipid) was added to 1.5 ml of PEG top phase and 1.5 ml of dextran bottom
phase in a 10 x 75 rom tube at 25°C. The contents were mixed by repeated
inversion for 1 min. Triplicate 50 ~l samples were removed for total
counts. The tubes were allowed to stand at 25°C for 25 min then triplicate
50 ~l samples of top phase and triplicate 20 ~l samples of bottom phase
were removed for counting. The difference between counts in top and bottom
phases represents counts at the interface; corrections due to adsorption
to the tube walls or at the air-water interface are negligible.
RESULTS AND DISCUSSION
Effect of vesicle Size and Composition
An essential difference between the partitioning of small molecular
weight solutes and particulates is that whereas solutes partition between
the two phases, particulates partition between one of the phases and the
2
microinter{aces between streams and droplets of the polymer phases as they
demix. This very fact that particles adsorb to the interfaces, means that
partitioning of particulates exhibits kinetics. As phase separation
proceeds progressively more particles are delivered to the bulk interface
until, at equilibrium, all particulates end up at the bulk interface
between the two phase-separated polymers. This clearly has important
operational significance with regard to choice of the appropriate time
point at which to sample the two phases.
In general terms a particle associates with an interface when the
change in surface free energy upon association with the interface is of
the same order or greater than the average thermal energy. If Brownian and
shear forces dominate, then the particle will be swept off the interface.
The change in surface free energy that occurs depends both on the
interfacial tension between the two polymer phases and also the surface
area of the absorbed particle, thus partitioning is sensitive to the size
of the particulate over a certain regime27.
100
f!! • • •
Z 80 "
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..0... .. f',.
Iii
0 40 ...
!Z ~
0
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(aJ: 20 0
w
Il.
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0 20 40 60 80 100 120
TIME (MIN)
Figure 1. The kinetics of the partitioning of EPC/EPG (6:4) vesicles in a
dextran T-500/PEG 6000 (5%/5% w/w) system containing O.llM
sodium phosphate pH 6.8. The filter pore size (nm) used for
sizing of the vesicles was (e) 50, (0) 100, (.6.) 200,
(.A.) 400 and (0) 600; (0) correspondstounsized
multilamellar vesicles. (Figure redrawn {rom re{.29)
The effect of vesicle size upon the kinetics of partitioning of
vesicles composed of egg phosphatidylcholine (EPC) and egg phosphatdyl
glycerol (EPG) (6:4 mole ratio) in a phase system of high interfacial
potential is shown in Figure 1. It is clear that there is no appreciable
difference in the rate at which vesicles (radiolabelled) are cleared from
the top phase for vesicles sized through 400 or 600 nm filters, or for
multilamellar vesicles (>1 micron diameter). For vesicles sized through
200 and 400 nm filters clearance to the bulk interface was slower and for
50nm vesicles, the majority of the vesicle (>80%) remained in the top
phase at 2 hr post mixing. Theoretical considerations indicate that for
phase systems with an interfacial tension of 5 x 10-3 erg/cm2, particles of
diameter less than or equal to approximately 30nm diamter will not adsorb
to the interface and so remain suspended in one phase28; consistent with
the results of Figure 1.
3
Albertsson's generalized application of the Br0nsted equation21
indicates that the partition of a particulate should be exponentially
dependent upon the surface area of the particulate and this is indeed
borne out by experimentation (Figure 2). The practical consequence of
these findings is that in order not to confuse size and charge effects it
is necessary to perform partition with model membranes of defined size29•
1.0
0
0
0.5 ~ •
l>.
...
0.0
:.:
.z... 0
•
.(1.5
·1.0
...
·1.5
0 0.5 1 1.5 2 2.5 3
SURFACE AREA (CM2x 109)
Figure 2. Plot of the natural logarithm of the partition coefficient K
versus vesicle surface area for EPC/EPG (6:4) vesicles sized
through 100, 200 and 400 nm pore size filters at (0) 13,
(.)25, (lI.)45 and(.)120 min after mixing. The partition
coefficient K is defined as (percent counts in top phase)/
(total - percent counts in top phase) .
Calibration curves for the partition of 100nm diameter unilamellar
vesicles containing dioleoyl phosphatidylcholine (DOPC) and various molar
ratios of dioleoyl phosphatidic acid (DOPA) and dioleoyl phosphatidyl
glycerol (DOPG) are shown in Figure 3. Consistent with Br0nsted theory,
there is an approximately linear relation between the logarithm of the
partition coefficient and the mole percent of charged lipid species in the
membrane, indicating the exponential sensitivity to charge. The region of
greatest sensitivity was for vesicles containing between approximately 2
and 6 mole percent of the charged species. In order to increase the
sensitivity of the phase systems to vesicles containing smaller amounts of
charged lipids, phase systems closer to the critical point (for two-phase
formation) can be used. Of course, for systems closer to the critical
point, other non charge-related factors such as the interfacial tension
may playa greater role in the partitioning. It is noteworthy that the
results of Figure 3 indicate that whereas the absolute value of the
partition coefficient differs for vesicles containing DOPA or DOPG, the
slope of the standard curves is very similar. One interpretation of this
is that the phase systems are equally sensitive to changes in surface
charge whether derived from DOPG or DOPA, but that the lipids are
differentially wetted by the lower dextran-rich phase. This increased
wetting as a function of surface charge should be manifested as a
decrease in contact angle; measurements upon systems containing egg
phosphatidylglycerol indicate that this is indeed the case (Figure 4).
Phase partition is sensitive to variation in surface charge over a
wide range as illustrated in Figure 5 for 100nm vesicles containing up to
4