Bioscience Reports, Vol. 9, No. 6, 1989
REVIEW
Membrane-Membrane Contact:
Involvement of Interfacial Instability in the
Generation of Discrete Contacts
W. T. Coakley 1'3 and D . GaHez 2
Received April 19, 1989
The classical approach to understanding the closeness of approach of two membranes has developed
from consideration of the net effect of an attractive van der Waals force and a repulsive electrostatic
force. The repulsive role of hydration forces and stereorepulsion glycocalyx forces have been recently
recognized and an analysis of the effect of crosslinking molecules has been developed. Implicit in
these approaches is the idea of an intercellular water layer of uniform thickness which narrows but
retains a uniform thickness as the cells move towards an equilibrium separation distance. Most
recently an attempt has been made to develop a physical chemical approach to contact which
accommodates the widespread occurrence of localized spatially separated point contacts between
interacting cells and membranes. It is based on ideas drawn from analysis of the conditions required to
destabilize thin liquid films so that thickness fluctuations develop spontaneously and grow as
interracial instabilities to give spatially periodic contact. Examples of plasma membrane behaviour
which are consistent with the interfacial instability approach are discussed and experiments involving
polycation, polyethylene glycol, dextran and lectin adhesion and agglutination of erythrocytes are
reviewed.
KEY WORDS: membrane contact; membrane instability; interfacial instability; cell adhesion; cell
agglutination.
1. INTRODUCTION
Early insights into the conditions which enable cells to remain as single cells in
suspensio n were based [4, 9] on the treatments available to explain the stability of
colloidal systems. In the DLVO approach [25] to colloidal stability it was
recognized that the long range van der Waals force would exert an attractive
force between particles in suspension. As the particles are drawn together an
electrostatic repulsive force between two particles becomes increasingly important
1 School of Pure and Applied Biology, University of Wales College of Cardiff, Museum Avenue,
Cardiff CF1 3TL, U.K.
2Service Chimie Physique, Universite Libre de Bruxelles, Campus Plaine, Boulevard du Tiomphe
1050 Bruxelles, Belgique.
3 To whom correspondence should be addressed.
675
0144-8463/89/1200-0675506.00/09 1989PlenumPublishingCorporation '
676
W.T. Coakleyand D. GaUez
and acts against the van der Waals force. The attractive and repulsive forces are
equal at a minimum ("the secondary Minimum") in the interactive potential
energy profile which identifies a stable equilibrium separation distance. If the
particles are forced closer together the repulsive electrostatic force would need to
be overcome but as the particle separation decreases towards zero the van der
Waals forces dominate again to give strong agglutination (at the "primary
minimum").
In addition to the above classical forces the development of reliable
force-measuring techniques since the late 1960's has revealed a variety of
interactions that depend on the nature of the surfaces and of the intervening
liquids [27]. By convention, the newer effects are classified as (i) solvation
("structural's or "hydration") forces which arise from the structuring or ordering
of liquid molecules when confined between two surfaces close together [40] and
(ii) repulsive entropic (steric or .fluctuation) forces which arise from the thermal
motion of protruding surface groups such as polymers (e.g. glycocalyx molecules
[3]) or lipid head groups. Thermal fluctuations of flexible lipid bilayers [17] give
rise to a repulsive interaction which may be included in this category [27].
It has been shown, for a number of liquids, that at separations greater than
ten solvent molecule diameters (i.e. >3 nm for water), classical van der Waals
and electrostatic interactions adequately describe the inter-surface forces [27]. It
is at lower separations that solvation forces become important. For the case of
biological bilayer membranes, with hydrophilic surface groups, these hydration
forces result in a short range monotonic repulsive force which decays exponentially with a characteristic length varying between 0.15 and 0.35 nm [7~ 26, 34].
In the case of the plasma membrane the presence of a macromelecular
glycocalyx of thickness of the order of 5-10 nm on many cells further complicates
the extent to which classical formulations can fully describe the interaction
between cells. The consequences, for the electrostatic energy of interaction, of
the volume distribution of cell surface charge within a glycocalyx has been
examined [13]. It has also been recognized that, in addition to stereorepulsion,
glycocalyx-specific effects such as osmotic force arising from the presence of the
glycoclyx molecules will exert a repulsion between adjacent cells [3]. Finally, the
knowledge that lectins and antigen-antibody interactions could overcome cell-cell
repulsion has led to the development of a number of attempts to account for
attractive molecular cross-bridging effects [3].
The recent appreciation of the presence of a wider range of interactions than
were considered when the DLVO theory was developed initially does not alter its
basic prediction of minima and maxima in the interactive energy profile between
adjacent particles. Implicit in the DLVO treatment of particle interaction is that
surfaces do not deform during approach and that, after reaching an energy
minimum, they maintain a uniform separation distance. In many animal cell-cell
interactions however and in most biological membrane-membrane interactions
the approaching surfaces are deformable and the post-contact separation distance
is not uniform. Close cell apposition, for instance, during fusion [33], cell
movement [52] or in some cases of polymer-induced adhesion often involves the
making of spatially discrete contact points separating regions where cell mem-
Interracial Instability and M e m b r a n e Contacts_
677
branes or a cell and its substratum are separated by distances which can be in
excess of 20 nm.
In an early application of DLVO theory to cell-cell interaction Curtis
proposed that the gap between the plasma membranes of cells in tissue represents
the secondary minimum separation and that regions of tight contact were
equivalent to adhesion in the primary minimum [9]. A proposed reduction in
electrostatic repulsion at the tips of microvilli ([37], critically reviewed by Curtis,
[9]) or at the tips of the pseudopdia of phagocytes [51] were early efforts to
explain how local regions of plasma membrane could penetrate the repulsion
maximum and make discrete local tight contact at a primary minimum.
Suggestions such as cell process formation however can only be possible solutions
to specific examples of the more general phenomenon of the co-existence of local
regions of close contact and of wider separation in a range of biological
membrane systems.
In the present paper we show how consideration of the fate of the narrowing
aqueous layer as two cells come together offers an explanation of why contact
between cells [19] or membranes [12] can be spatially periodic. We review
examples of spatially periodic contact induced by a number of polymers during
adhesion of erythrocytes and we also discuss some examples of spatially periodic
contact formation in cell biology. The paper is part of a search (i) for
understanding of the physical chemistry of the occurrence of spatially periodic
close apposition in cell biology and (ii) for development of an informed basis for
the selection of enzyme treatments which modify the cell surface and for the
selection of non-physiological chemical or physical conditions where close local
contact is required during biotechnological exploitation of cells, as in cell fusion.
2. T H E O R Y
(a). The first general question to be answered is : What is the equilibrium
distance between two charged cells? This question can be considered for the case
of cell-cell contact by using classical DLVO theory [25, 54] modified at short
distances of approach by new repulsive interactions. It is supposed in this theory
that the two surfaces remain fiat during their approach and that the approach is
slow so that the system is clearly at thermodynamic equilibrium.
The potential energy of attraction per unit area, Vw, due to the long range
van der Waals force between two parallel blocks of infinite extension is given [25]
by
gw = -A.
(127~h2) - 1
(1)
where h is the separation between the parallel surfaces and A is the Hamaker
constant. For two approaching cells, h may be calculated as the distance between
the lipidic parts of the two membranes. For the aqueous intercellular layer the
order of magnitude of the Hamaker constant is taken as 5 9 10 -21 J [36].
678
W.T. Coakleyand D. GaUez
The repulsive electrostatic potential energy per unit area, due to the overlay
of the double layers, may be evaluated directly from DLVO theory for a charge
density located at the surface of the plates. However in the case of cellular
surfaces, the charge is distributed within the surface coat (i.e. the glycocalyx).
The electrostatic potential energy then reads [13]
VE = (a2o/(eeok)(1 - tanh (o:)]/[(1 + fl)(fl + tanh (or))]
(2)
where the surface charge density a0 is 0.02 Cm -2, e is the relative permittivity of
water (e = 80), e0 is the permittivity of free space; a~= k(h/2 - 6) where k is the
inverse Debye length, 6 is tbe width of the glycocalyx (6 = 5 nm and fl = kdi.
Adding the attractive and repulsive terms of Eqn. 1 and 2 for cells in
physiological medium gives the usual DLVO energy profile with a secondary
minimum at a separation of about 15 nm.
However, even in the equilibrium case, the potential energy profile must be
modified since, at short distances of approach, cells experience very high
repulsive steric and hydration forces [3]. For interacting cells the steric forces
arise because the cell membranes are coated by a hydrated layer of long-chain
polymers (the glycocalyx) which overlay at short distances of approach (h < 26).
Additional repulsive energy terms must then be introduced f o r the solvent
exclusion and polymer compressibility [3], of the form
VR = PRJ.R exp (--h/)~a)
(3)
where ~R and PR a r e taken as 3 nm and 105N m -2 respectively for a glycocalyx
thickness of 5 nm. The uncertainty in these paramenters may be as much as an
order of magnitude [19].
Finally if some specific molecules (e.g. lectins) or polycations such as
polylysine are present attractive polymer crosslinking is also possible and will
overcome the cell-cell repulsion at short distances. This will give rise to an
additional attractive potential energy
VA = PAZA exp (--h/).g)
(4)
where ~A and PA a r e taken, for example for polylysine, as 4 nm and 4.104 N m -2
respectively [19], again for a glycocaclyx thickness of 5 nm.
Figure 1 shows the attractive and the repulsive components of Eqns. 1-4 and
the potential energy VT = Vw + VE + VR + VA. The continuous curves (Vw and
VE) correspond to the usual DLVO theory while the interrupted curves (VR and
VA) are new contributions. The total interaction energy VT has the classical form
of the interaction energy presented for two cell surfaces i.e. a net long range
attractive effect at large separation, a secondary minimum when attractive and
repulsive forces cancel (point a), a repulsive maximum and a primary minimum
(point a'). Weak cell adhesion may occur in the secondary minimum (around
15 nm) while it has been customary to view tight cell-cell contact as arising
through contact in the primary minimum (around 5 nm).
(b) The second important question to discuss is now: Why, under some
conditions, the contact between cells appears to be a spatially periodic phenomenon? To answer this question a dynamic theory is clearly needed, since the system
Interfacial Instability and Membrane
Contacts
679
_
-t //l',.
,5-
%
g
\
\.\
'\
16 18-.20 22 2~
O'
I1
L
//
'
-S
l II
I"
i!
/t
-10 -
i!
/
Fig. 1. The potential energy per unit area between
two plane plasma membranes due to (i) - long
range van der Waals force; (ii) - electrostatic
repulsion; (iii) . . . .
glycocalyx stereo-repulsion and
(iv)
macromolecular cross linking. The net
change, with membrane separation, in the sum of the
four terms (curve a', d, c, a, b) is also shown.
is in out-of-equilibrium conditions. The movement of the intercellular fluid must
be considered as well as the movement of the two cell surfaces. The model
adopted to describe the system is the following.
The narrowing water layer between two cells which are drawing close
together and the plasma membranes of the two cells may be considered as a thin
fluid film between the membranes of the two cells. In the general linear analysis
of the stability of interfaces the effect of small perturbations of the interface shape
due to thermally or externally induced fluctuations is considered (Fig. 2). The
deformation of a plane interface can then be expressed as a Fourier integral of all
the possible wavelengths of the perturbation. If the component of the displacement associated with each of the wavelengths decreases with time the system will
be stable. If the displacement associated with any wavelength grows the system is
unstable. The unstable interface may reach a new steady state with interface
deformed but intact or it may breakup to form droplets. A film may become
unstable through development of a bending wave, where the film deforms
(without thickness fluctuation) through exponential growth of the amplitude of a
surface wave at right angles to the plane of the undisturbed thin layer.
680
W. T. Coakley and D. Gallez
~~~\x!/
/"
h
cei2l
Fig. 2. Two interacting cells separated by an
aqueous film of width h.
Alternatively the layer may develop periodic thickness fluctuations (a squeezing
wave) as in Fig. 3.
The stability criterion for bending wave growth is:
aT > 0
(5)
aT = 2as + OW + OE ~- OR -~- OA
(6)
with
where 2as is the pure surface tension at the two surfaces of the layer, Ow is the
tension due to the long range van der Waals forces, OE is the tension due to
electrical forces and OR and OA are the tensions due to stereo-repulsion and
macromolecular cross-linking respectively [19]. Spontaneous growth of spatially
periodic bending waves (in times of ls or less) leading to vesicle formation has
indeed been recorded by cinemicroscopy for isolated single erythrocytes whose
elastic properties were modified by thermal denaturation of the membrane
skeleton protein, spectrin [4, 8].
The stability criterion for the squeezing wave growth is:
kaOT -- 2dC~T/dh > 0
(7)
were k is 6.28 times the inverse of the wavelength of the perturbation and the
total disjoining pressure (the first derivitive of the potential terms in Eqns. 1-4) is
.TICT = JgW -[- J~'E -~- '~R "~ ~ A [19]. T h e i n d i v i d u a l f i r s t d e r i v a t i v e s a r e given by
d:rw/dh = A/2(2~Th4)
dJrE/dh = [(oo2k)/(geeo)][Sech 2 (cr)]{[fl + tanh (a0].
[tanh (if)] + 2Sech 2 (a0}/[fl + tanh (c~)]3
d~R/dh = --PR exp (--h/~,R)/~-R
(8)
(9)
(10)
d:rg/dh = PA exp (--h/~,g)/),A
(11)
InterfacialInstabilityand MembraneContacts
681
o) Bending Hode
b) Squeezing Mode
Fig. 3. The two modes of vibration for symmetrical
systems (a) The bending mode BE with in-phase
transverse displacements v; (b) the squeezing mode
(SQ) with 180 out of phase transverse displacements
and in phase longitudinaldisplacementsDv.
For separations in excess of 18 nm (point b) the dominant contribution to the
derivative dazT/dh is destabilising (van der Waals attraction). Point b corresponds
to a change in the curvature of the total energy potential V; at this point the
contribution dJrT/dh changes sign (from positive to negative and the system
becomes stable, in accordance with criterion (7). This means that small
fluctuations around the secondary minimum (point a) will decay. As the
separation, h, decreases further the repulsive steric forces become dominant and
the system would remain stable in the absence of attractive cross-bridging
interactions. Between points c and d the system may again become unstable
(d~rT/dh >0) due to short range attraction, and small fluctuations will grow,
leading to periodic point contacts (we suppose in that case that the close contact
occurs in the first minimum, around 5 nm). In other words this means that if the
system is lead to a region of instability by an external constraint (polylysine
treated cells, for instance), the SQ wave will grow between the cells leading to
spatially periodic intercellular contact points. The dominant instability wavelength may be obtained [19] by taking the nondimensional wavenumber (kh) for
which the rate of growth of the instability is maximum. This wavenumber (kh) is
typically of the order of 0.2 so that, for a cell separation of about 20 nm, the
dominant wavelength ranges around 0.6/,m for polylysine treated erythrocytes
(Fig. 4) which is the same order of magnitude as the average spacing between
contact regions of the periodic patterns. This dominant wavelength is independent of the viscosity (see Fig. 4) but depends on the physico-chemical parameters
of the cell membrane and of the intercellular layer.
The theory also predicts the time for development of the instability, which is
682
W . T . Coakley and D. Gallez
W (x 30-?see-I}
3C
i0-2p
20
0
J -
lp
10
20
30
kh(xlO-~'}
Fig, 4. The rate of growth as a function of the dimensionless wave
number kh for two interacting erythrocytes, for several values of the
viscosity of the intercellular aqueous layer (19).
of the order of 0.1 s (for an intercellular layer of viscosity about 1 Poise, due to
the presence of the glycocalyx); this can be compared with the time (0.3 s) for
spherical doublet formation [46]. This first evaluation of the dynamic parameters
of the system is then in good agreement with the existing experimental
observations.
EXPERIMENTAL
(3a) Studies of Polymer Adhesion of Erythrocytes
Recent studies of erythrocyte-erythrocyte adhesion and agglutination induced by polycations [6, 24], polyethylene glycol [46], dextran [5] and the lectin
wheat germ agglutinin [11] have provided insights into the development of local
contacts. Cell contact regions have been examined by light microscopy in
chemically fixed and in unfixed cells and by electron microscopy in chemically and
in cryo-fixed cells. The time scale involved in cell adhesion has been determined
by measurements of the rate of adhesion of cells observed by video microscopy as
they came together.
3a(i) Erythrocyte adhesion in solutions of polycations. A number of studies
of the adhesion of erythrocytes by polycations e.g. polylysine [6, 23, 24, 31],
polyarginine [24,31], polyornithine, polyvinylamine, polyvinylpiperidine and
Interfacial Instabilityand Membrane Contacts
683
Fig. 5. (a) Light micrograph of erythrocytes glutaraldehyde-fixedfollowing exposure to
20/tg/ml polylysineshowingcontact pattern (b) two erythrocytes(as in (a)) with a small region
of cell overlap. (c) Light micrograph of an unfixed cell clump showing contact detail between
cells washed once after exposure to 10 #g/ml polylysine;(d) Transmissionelectron micrographs
of cells treated as in (a); (e) Freeze fracture electron micrograph of cryo-fixedcells showing
three contacts separated by distances of less than 1 ~m; (f, g) Scanningelectron micrographsof
cell contact surfaces exposed by shearing glutaraldehyde-fixedcell clumps ((a-d),f[6]; (e) [24];
(g) [23].
protamine [31] have been published. For each polycation tested the threshold
concentration for adhesion decreased with increasing molecular weight. For
polymers of the lower molecular weight basic subunits the threshold concentration for adhesion was close to the concentration at which the polycation began to
modify the electrophoretic mobility of the erythrocyte [6, 31].
Erythrocytes fixed with glutaralehyde following exposure to polylysine show
a regular pattern of contacts at the intercellular seam (Fig. 5a). The pattern
occurs only where the cells overlap (Fig. 5b). Periodic contact spacing could be
seen by light microscopy of unfixed cells (Fig. 5c) and by freeze fracture electron
microscopy of cryo-fixed cells which had not been exposed to chemical fixative
(Fig. 5e). Transmission electron micrographs of polycation treated cells also show
regular spacing of contact regions between cells in a large clump (Fig. 5d). The
wavy profile is confined to the regions where cell surfaces are opposite each other
i.e. the erythrocyte membrane exposed on the outside of the clump shows no
undulation. Scanning electron micrographs (Fig. 5f, g; where the contac,t surfaces
have been exposed by stress) confirm that the pattern is confined to discrete
regions of the cell surface. The average distance between contact points in
polylysine treated cells was 0.83/~m [6].
Light and electron microscopy of polycation induced cell contact was first
examined by Katchalsky et al. [31]. These authors found interruption of contact
but made no comment on spatial periodicity between contact regions. Hewison
[23] showed that periodicity of cell contacts occurred but was not widespread in
cells fixed in 0.5% osmium tetroxide (the sole fixative used by Katchalsky et al.
684
W.T. Coakleyand D. Gallez
Fig. 6. Transmission electron micrograph of
erythrocyte-yeast adhesion in 25 #g/ml 240 kDa polylysine showingspatially periodic nature of cell contact [24].
[31]). However periodicity was widespread when cells were fixed solely in low
concentrations of osmium tetroxide (0.05%-0.005%) suggesting that the lack of
comment on spatial periodicity by Katchalsky et al. (1959) was due to the higher
concentration of osmium tetroxide used in that work.
3a(ii) Polycation induced erythrocyte adhesion to a solid (yeast) surface:
When two erythrocytes suspended in a polycation solution come into contact the
surfaces of both cell surfaces bend as the contact pattern is formed. The adhering
surfaces have symmetrical electrical and mechanical properties. When surfaces
with asymmetric properties are brought together by polycation s a pattern also
develops but the wavy profile is confined to the less rigid erythrocyte surface.
Figure 6 shows a wavy erythrocyte-yeast (Schizosaccharomyces pombe) profile
with the erythrocyte forming pseudopodiaqike extensions on the yeast surface
[24].
3a(iii) Erythrocytes in polyethylene glycol: Exposure of cells to concentrations of polyethylene glycol (PEG) in range 45-50% is a step in the chemical
fusion of erythrocytes and of other cells. The freeze fracture electron micrographs
of Knutton [33] show local membrane contact points separated by distances in the
range 0.2/~m-0.8/~m in erythrocytes which has been exposed to 50% w/v PEG
solutions. A transmission electron micrograph [1] of human erythrocytes which
were fusing after suspension in 40% PEG and resuspension in buffer showed two
examples of arrays of 0.2/~m spaced vesicles at the fusing cell junction. Light
micrographs of erythrocytes fixed after suspension in 25% PEG show occasional
examples of arrays of intercellular spaces separated by distances of the order of a
micron [46] while transmission electron micrographs of such cells shows widespread evidence for an additional smaller spacing of the order of 0.4/~m between
contacts (Darmani, personal communication). Further work is required to
establish the dependence of both prefusion contact-point and fusion site
separation on experimental conditions.
3a(iv) Erythrocytes exposed to lectins: Lectins are identified as a class of
biopolymers by their ability to agglutinate erythrocytes through interaction with
glycocalyx carbohydrate sugar residues. Despite the ubiquity of lectins in nature
and the many applications they find in biology there appears to be only a small
number [11, 15, 16, 21] of published studies of the energetics and dynamics of
lectin agglutination of erythrocytes. Recently Darmani et al. [11] investigated the
Interfacial Instability and Membrane Contacts
685
Fig. 7. Contact seam of cells exposed to (a) 2% dextran T500 (b) 4% dextran (c) 4% dextran
followingpronase pretreatment of cells; (d) 20/~g/ml WGA [5].
time dependence of agglutination of human erythrocytes by wheat germ
agglutinin (WGA). They found that most cells had agglutinated within 5 min of
initial exposure to lectin. This early agglutination however occurred almost
exclusively at cell edges. As time of exposure progressed the area of contact
between individual cells grew and the size of the agglutinate increased. This
contact spreading phenomenon developed by the making of periodic cell contacts
separated by distances of the order of 0.8 ~m (Fig. 7d). The rate of contact
spreading could be increased 30-fold by continuously agitating the cells during
incubation. It was argued that the intercellular water layer at the contact edge
became narrow during agitation and the narrow layer became unstable to give
periodic contacts as in Fig. 7d. Neither W G A receptor labelling or freeze-fracture
electron microscopy provided any evidence of precontact lectin-induced spatially
periodic concentration of receptors in the plane of the erythrocyte membrane
[111.
Spatially periodic cell contact patterns were also resolved by light microscopy
in unfixed cell agglutinates after 15 min on a microscope slide [11]. Internalization
of plasma membrane vesicles of diameter of the same order as the inter-contact
spacing occurred with prolonged incubation of erythrocytes in WGA. This
internalization may have the same origin as the internalization process exploited
by coating mammalian cell (rat embryo neurocrest) surfaces with W G A in order
to effect vesicular transport of horse radish peroxidase to the Golgi apparatus
[45]. The fact that membrane internalization by vesiculation occurs supports the
conclusion that in this case discrete rather than continuous cell contact is achieved
by WGA treatment. It has also been reported that addition of W G A to mouse
embryo culture medium causes increased apposition of two cell blastomeres. This
is followed by the formation of numerous small intercellular spaces [30]
necessarily implying local contact formation.
3a(v) Cells in dextrans: In contrast to the situation for polycations, PEG and
WGA there is little evidence of periodic cell contact when normal human
erythrocytes adhere in the presence of dextran. Electron microscopy shows
continuous parallel-surface seams in cells treated with 3% and 4% solutions of
75,000 MW dextran [29, 44]. Figure 7(a) similarly shows that the cell surfaces
within dextran (2% w/v; 450,000 MW) induced aggregates remain parallel [5].
However spatially periodic contact was observed when cells in 4% dextran T500
were pretreated with pronase (Fig. 7c). The uniform separation of control
686
w.T. Coakleyand D. GaUez
erythrocytes in 2% dextran (Fig. 7a) is consistent with contact formation in a
secondary minimum (Fig. 1) based on the equilibrium DLVO approach while the
periodic contact induced in cells whose glycocalices have been enzymically
pretreated (Fig. 7c) is consistent with the formation of contact by an out of
equilibrium process. Uniform separation and discrete periodic contacts have
recently been confirmed for control and pronase treated cells respectively when
both cell suspensions were in 2% dextran T500 (Darmani, personal communication). The lateral separation of contact points depends on the extent of pronase
pretreatment in a manner in accord with interfacial instability theory.
3a(vi) The time scale of erythrocyte adhesion by polymers: Tilley et al. [46]
developed a technique for examining cell adhesion in suspension. Cells in
rectangular microcapillaries were levitated in an ultrasonic standing wave field
and were concentrated close to pressure nodes in the sound field, where cell
interactions were video recorded. Cells in low (0.5%-1.5% w/v) concentrations
of dextran T500 came together, interacted and formed a rouleau within 2.5 s-17 s
of making initial contact. Following initial contact of cells in 5% and 7% dextran
there was a pause (15 s) before the "cell pair" began a process of rapid (2.7 s)
mutual engulfment which resulted in the formation of a spherical cell doublet or a
convex-ended cell clump. The pause and engulfment times for cells in 20/tg/ml
polylysine were 2.7 s and 0.3 s respectively [45]. Cells in 25% w/v PEG stayed in
contact for a few minutes before beginning an engulfment stage which lasted one
second, van Oss and Coakley [50] suggested that the underlying process in the
dextran, polyethylene glycol and polylysine formation of cell doublets is a phase
separation effect in which, in the case of dextran, the sugar polymer coating the
erythrocyte glycocalyx were repelled (because of monopolar repulsion [49]) by
the dextran molecules in solution. In the case of polycations the repulsion
between the polycation coated glycocalyx and the polyelectrolyte in solution
provided an electrostatic repulsion to bring about phase separation. Osmotic
effects due to partial dextran depletion in the narrow water layer between cells
may also contribute to the cell-cell attractive force during the final engulfment
stage. The rapid mutual engulfment phase seen in polylysine, dextran and PEG
treated cells is not a feature of WGA-induced cell agglutination [11]. Spreading of
cell contact in the latter system appears to require that membranes of cell pairs be
brought into close contact by shear stress due to fluid currents in the suspending
phase.
The time scale of contact spreading in polylysine gives an upper limit on the
development of membrane contact. The fastest time observed experimentally was
with polylysine induced cell adhesion where the spherical doublet was formed
within 300 ms [46]. This time is comparable to that predicted for wave growth
where the viscosity of the thin film is taken (because of glycocalyx molecules) to
be about about 1 Poise (Fig. 4).
(3b) Local Contact Formation in Living Systems
3b(i) Vesiculation during acrosome reaction: Spatially discrete contact
formation is widely observed in cell-cell and membrane-membrane interactions.
Interfacial Instability and Membrane Contacts
687
Local contact of the apposing faces of the acrosome outer membrane and the
plasma membrane of boar spermatozoa during the acrosome reaction leads to the
production of a sheet of essentially equally sized hybrid vesicles (implying
regularly spaced contact/fusion sites) consisting of hemispheres of both membranes [42]. The contact separation in the fusing boar sperm membranes is about
0.25/~m [42]. Phospholipase A activity is detected prior to the acrosome reaction.
Meizel [35] pointed out that action of the phospholipase would leave a fusogenic
residue in the bilayers. Addition of sea urchin sperm to sea water containing egg
jelly triggers a calcium influx and initiates the acrosome reaction [22, 47].
Vesiculation of the sperm head membrane is one of five distinct stages seen in sea
urchin Sperm fixed within 1 s of initiation of the acrosome reaction [10]. This
observation suggests that vesiculation can occur in a small part of one second.
3b(ii) Cell locomotion and spreading: The distribution of cell-substratum
contact points during locomotion of the large amoeba Chaos chaos on glass is
"regular" [2]. Interference reflection microscopy shows point "focal" cellsubstratum contact development during locomotion of the small soil amoeba
Naegleria gruberi [38] over a glass substratum. The focal points formed during
Fig. 8. Electron micrograph of hybrid vesicles formed from the
apposing faces of the acrosome outer membrane and the plasma
membrane of boar spermatozoa during the acrosome reaction.
The acrosomal membrane part of the hybrid is identified as the
more fuzzy hemisphere (f). The arrows show the join between
the two hemispheres is in register between adjacent vesicles [42].
688
W.T. Coakley and D. Gallez
Naegleria gruberi locomotion emerged only from broad platform regions of
'associated contact" (occupying about 1/3 of the cell cross-sectional area)
between the plasma membrane and glass. When focal contacts were formed they
acted to stabilise the area of associated contact otherwise undulations of the
plasma membrane caused the location of the regions of associated contact to
change faster than the temporal resolution (2 s) of the photographic system
employed [32]. The separation distance of the "associated contact" platform from
the glass substratum depended strongly (110 nm in distilled water and 20 nm in
3 mM NaCI) and reversibly on the ionic strength of the cell's environment [38].
King et al. [32] gave an average value of one contact per 5.6/~m 2 (equivalent to
an average focal contact separation of 2.4/~m) for cells in distilled water. Values
of h = 110 nm and Z = 2.4 #m give kh = 0.28 for amoeba in distilled water. This
value is of the order of the kh parameter for squeezing wave growth between two
cells having the surface properties of polylysine treated erythrocytes in physiological saline (Fig. 4). It would be expected [39] that the value of kh for which o9 is a
maximum would show some dependence on ionic strength. In a careful review of
the results of focal point distribution King et al. [32] favoured the conclusion that
a significant increase in the number of focal contacts per region of "associated
contact" occurred for smaller gap distances. The average spacing between focal
contact points was reduced to 1/~m when cells moved over a polylysine film at
a separation of less than 20 nm.
Metazoan cells display a wide variety of structurally defined cell contacts,
differing in their dimensions, spatial interrelationships, temporal development
and physiological role [20]. The formation of new dot contacts (0.1-0.2/~m
diameter) is usually restricted to the sites of undulatory [28] pseudopodial activity
in spreading fibroblasts [52, 53]. These dot contacts are not associated with large
actin bundles but some of the contacts later mature and become associated with
the microfilament bundles characteristic of developed focal contacts [52]. Segel,
Volk and Geiger [43] describe the initial development of small radial patches
("contact spots") of nascent focal contacts adjacent to a belt of "close contact")
at the periphery of spreading chicken gizzard cell. Later the cells had developed a
spatially periodic pattern of radial focal contacts. The authors comment that any
theory of cell contact and spreading must explain the observed periodic
structures. They suggest that the periodicity may arise from a reaction-diffusion
instability where the formation of one ceU-substrate bond at a point makes it
more likely that other binding molecules will duster in the neighbourhood. An
expression was derived but no estimates were made of a characteristic distance
for a spatial periodicity of clustered binding sites based on a molecular
concentration instability. In the case of initial contact formation an important
difference between the approach of Segel et al. [43] and that of this paper is that
the former approach would imply a spatial distribution of concentrated regions of
adhesion complexes (as putative contacts) which necessarily occurs after a single
contact is made. The mechanical instability approach on the other hand considers
the spatial periodicity of contact points to arise because molecular contact would
be most likely to occur at the spatially periodic locations where membranes
approach each other or a substratum most closely. The mechanical model allows
Interfaciai Instability and Membrane Contacts
689
that the initial contact points may grow or be reinforced by a positivecooperativity mechanism (as in fibronectin receptor concentration at focal points
of stationary vertebrate embryonic cells [14] or that the distribution of initial dot
contacts can subsequently be modified (through chemical reorganisation of the
cytoskeleton) to give large, more widely spaced, focal contacts [53]. Experimental
evidence gives some support to the mechanical model (no precontact concentration of binding molecules) in the particular case of amoeboid focal contact
formation where, in distilled water, the focal contact region begins its journey
towards the substratum from a distance of over 100 nm [38]. In addition Geiger et
al. [20] state as their general finding that the molecular assembly of the adherens
junction structure in tissue cells is initiated by the contact itself.
Predator-prey contact during phagocytosis is regarded as a particular case of
cell (predator) spreading on a substrate (prey). Electron micrographs of yeast
ingestion by cultured human vascular endothelial cells [41] show a regular contact
separation distance.
3b(iii) Prefusion cell contact: In addition to the comments on periodic
contact during PEG-induced erythrocyte contact and fusion above it has been
reported that when plant protoplasts are drawn together by dielectrophoresis and
are then exposed to a fusogenic high voltage pulse [55] small vesicles are
produced at the cell junction and there is a net loss of external surface
membrane. A regular spacing of about 0.8/tm can also be seen in the electron
micrographs of the contact region of erythrocyte ghost membranes fusing in the
presence of calcium phosphate [56].
CONCLUSIONS
The suggestion above associating the formation of periodic membrane
contact with surface wave development requires that the cell boundary involved
should not be very stiff and that the surface tension of the cell should be low. In
addition to the rapid undulations which occur in the amoeboid ventral surface and
at the margins of spreading mammalian cells it is known that erythrocyte cell
thickness undergoes rapid (a few Hertz) transient variations (erythrocyte flicker)
of amplitude up to 200 nm. The damped surface wave associated with such
thickness fluctuations has a wavelength of about 0.6 ~m [18].
The experimental evidence considered above (Fig. 7) for the parallel uniform
separation of the surfaces of adjacent cells in dextran and for laterally periodic
contacts between pronase treated cells in dextran suggest conditions where
contact formation switches from an equilibrium separation which may be
described by the classical DLVO treatment to a non-equilibrium transition
requiring a dynamic theory. The dependence of lateral spacing on the extent of
pronase treatment suggests that quantitative tests of the theory may be possible in
that system. The results for polylysine, PEG and lectin induced contact suggest
that the nonequilibrium transition is not uncommon in polymer induced adhesion
of erythrocytes. The observation of spatial periodicity of contact in other
biological systems suggest that the growth of an interracial instability may
W . T . Coakley and D. Gallez
690
underlie the physical chemistry which allows local point contact to occur where
otherwise increased attraction would lead to narrowing, but uniform, separation.
ACKNOWLEDGEMENTS
DG would like to thank the Belgian Government (ARC) for support for this
research. WTC and DG are grateful to the EEC for a Stimulation Action Grant
(Ref.: 85200151 U.K. 108UJUI).
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