Bioscience Reports, Vol. 7, No. 4, 1987
Molecular Mechanisms of Membrane Fusion:
Steps During Phospholipid and Exocytotic
Membrane Fusion
Joshua Zimmerberg
Received July 7, 1987
KEY WORDS: exocytosis;fusion,membranefusion; phospholipid.
Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore
widening, and content discharge. Experiments on both synthetic and natural
membranes are presented to show each of these steps. Major differences are seen in the
two fusing systems. These differences are discussed in terms of molecular mechanisms
of fusion.
BACKGROUND
Exocytosis is a ubiquitous biological phenomena which includes a physically unique
sl;ep: membrane fusion. We can easily study surface coalescence of liquids. Droplets of
water, oil, mercury, and bubbles will readily combine upon contact to form a larger
drop. But solids usually do not fuse. Two red balloons do not suddenly become one
upon contact, regardless of the force with which they touch or are brought together.
Very clean metal surfaces will adhere with instantaneous delocalization of electrons,
but not rapid diffusion of nuclear lattice positions. Liquid films, such as soap or milk
bubbles in air will fuse and lyse if stimulated with electrical charge (Boys, 1959). All of
these fusing systems, have in common surfaces which can freely change their area. In
contradistinction, biological membranes have a fixed surface area which can withstand
little stretch (Kwok and Evans, 1981). They are finite-width structures, a fluid yet
ordered array, separating aqueous liquids. Yet biological membranes fuse in
physiologically desirable ways to effect macromolecular secretion, neurotransmitter
PSL, DCRT,and LBM,NIDDK, Bldg. 12A,Rm 2007,NationalInstitutesofHealth, Bethesda,MD 20892,
USA.
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0144-8463/87/0400-0251505.00/09 1987 Plenum Publishing Corporation
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Zimmerberg
release, protein sorting, membrane insertion, and intracellular trafficking (c.f. this
volume). The molecular forces, mechanisms, and components driving, actualizing, and
catalysing fusion are still obscure.
We have approached the study of biological membrane fusion by first studying
synthetic membranes composed of biological material, then applying results of that
study to define hypotheses for biological membrane fusion. In this article I will discuss
these and other results to understand the differences and similarities between
phospholipid bilayer and biological membrane fusion. In this way, we might guess the
role of the other cellular constituents as additions to bilayers in fusion. From
comparison of bilayer and natural systems, we tend to believe that fusion of
phospholipid membranes is not the same molecular event as biological membrane
fusion and that specific biochemical steps should be proposed.
It is useful to break up exocytosis into a sequence of component steps leading to
transport of large molecular weight substances from the insides of granules to the
extracellular space:
1. Adhesion: intimate contact between the two membranes that are to fuse, and
dehydration of the inter-membrane space (Fig. lb).
2. Fusion/pore formation: molecular mixing and rearrangement leading to a
connection of the granule interior with the extracellular space (Fig. lc). Topological
continuity from inside of the secretory vesicle to the external surface of the plasma
membrane.
4. Pore widening: a further opening up of the exocytotic pore (Fig. ld).
5. Discharge of the contents to the outside: once the exocytotic pore is larger than
the secretory molecule (Fig. ld,e).
Typically, one measures experimentally one or two of these events, and takes that
measurement to represent all of exocytosis. However, the molecular constraints on
t,i'ii',ii',iii
a
b
c
d
e
Fig. 1. A drawing of the events thought to occur in exocytotic membrane fusion. The membranes (a)
must first adhere (b), then fuse with pore formation (c). This pore must widen (d) to allow content
discharge (e).
Molecular Mechanisms of Membrane Fusion
253
mechanism are quite different for each step and will be considered individually. In this
article I will consider these steps in different membrane fusion systems.
PHOSPHOLIPID MEMBRANE FUSION
Theories derived from measurements of forces leading to adhesion, molecular
rearrangement, pore formation and content discharge in phospholipid bilayers can
later be compared to experimental observations of biological exocytosis. Both vesiclevesicle and vesicle-planar membrane fusion have been documented and have been
recently reviewed (Finkelstein, Zimmerberg and Cohen, 1986; Rand and Parsegian,
1986; Wilschut and Hoeckstra, 1987). The system akin to exocytosis is the fusion of
spherical phospholipid vesicles with planar phospholipid membranes (Zimmerberg,
Cohen and Finkelstein, 1980; Cohen, Zimmerberg and Finkelstein, 1980).
Adhesion-Dehydration
The first step is membrane contact. Given the low concentration of free monomer
of phospholipids (~ 10- lz M) in equilibrium with lamellar phase, membrane merger
cannot occur across aqueous media (Tanford, 1973). Either lipid or protein must
achieve molecular contact. If the surfaces are initially hydrated, then this will be a
dehydration reaction. Dehydration of macromolecular and membrane surfaces has
been studied in some detail (Parsegian, Rau and Zimmerberg, 1986; Parsegian, Rand
and Rau, 1986; Niles and Cohen, 1987b). With x-ray diffraction, one can measure the
distance between molecular surfaces (DNA, phospholipid membranes, carbohydrate,
protein) as a function of osmotic stress. Direct measurements of the forces required to
push two phospholipid bilayers together led to the discovery of hydration forces
(LeNeveu, Rand and Parsegian, 1976; Parsegian, Fuller and Rand, 1979; Rand and
Parsegian, 1986). Similar experiments can be performed on DNA rods (Rau, Lee and
Parsegian, 1984). The same kind of exponentially-growing repulsive force has been
measured with DNA as with membranes. In both cases, this hydration force is the
predominant barrier to molecular contact. Recently an attractive form of the
hydration force has been recognized (Rau and Parsegian, 1985; Rau and Parsegian,
1986). DNA will suddenly dehydrate and collapse upon the addition of, for example,
manganese at specific values of temperature and pressure. It has been postulated that
the sudden collapse of phosphatidylserine membranes upon addition of calcium is a
reflection of a positive or attractive component of the dehydration (Parsegian and
Rand, 1983), although fluctuating coulombic forces may also play a role (Marra, 1986;
Guldbrand, Jonsson, Wennerstrom and Linse, 1984).
In the fusion of phospholipid vesicles to planar phospholipid membranes the first
observable step is prefusion adherence (Finkelstein, Zimmerberg and Cohen, 1986).
There are two ways of detecting the prefusion state between vesicle and planar
membrane. One is to fail to separate vesicles from a planar membrane despite vigorous
washing of the aqueous phase that once contained vesicles (Akabas, Cohen and
Finkelstein, 1984; Cohen, Akabas and Finkelstein, 1982). The second is to observe
vesicle adherence directly by fluorescence microscopy (Niles and Cohen, 1987b; Niles
254
Zimmerberg
and Cohen, 1987a; Woodbury, 1986c). The adherent state is a prerequisite for any of
the further steps in fusion. The proximity of contact (< 5 A) can be deduced from the
fact that water flux across two bilayers causes fusion (Akabas, Cohen and Finkelstein,
1984). The structure of this pre-fusion state is not known. The tight, dehydrated
apposition of phosphatidylserine membranes in the presence of calcium may reflect a
cross-linked region arising from attractive hydration forces (Portis, Newton,
Pangborn and Papahadjopolous, 1979).
Molecular Rearrangement-Pore Formation
The next event to consider after adhesion is fusion: the molecular transformation
and rearrangement that merges the surfaces allowing surface continuity and pore
formation between the granule interior and membrane exterior. In vesicle-vesicle
fusion, lipid mixing can be used to detect fusion, but this can be difficult to differentiate
from non-fusional exchange (Martin and MacDonald, 1976). In the case of vesicle to
planar membrane fusion, the fusion pore can be detected electrically (Fig. 2). One can
easily measure the current that passes across the planar membrane to obtain its ionic
permeability. If we put channels (proteins which make the vesicle very permeable to
ions) into a vesicle, then we can measure fusion as the incorporation of those channels
into the planar membranes. As soon as a conduit for ion flow is established across the
planar membrane during fusion, one could see a current proportional to the
conductance of that conduit (Fig. 2). In fact, when one puts phospholipid vesicles on
one side of the planar bilayer, under appropriate conditions one does see simultaneous
insertion of channels from vesicle into planar phospholipid membrane (Cohen,
Zimmerberg and Finkelstein, 1980; Cohen, Akabas, Zimmerberg and Finkelstein,
1984). In this case the resistance to current flow would be a sum of two series
resistances: the resistance of the vesicle membrane and the pore resistance (Fig. 2b).
Since we see the full step in current within 200 #s (Cohen, Zimmerberg and Finkelstein,
1980; Cohen, Akabas, Zimmerberg and Finkelstein, 1984), we know that within 200 #s
the resistance to current flow contributed by the exocytotic pore is insignificant. In
other words, the initial pore which forms during fusion of phospholipid vesicles to
planar phospholipid membranes opens up to a relatively large size almost
instantaneously. This size can be imagined by calculating the diameter of a cylinder of
sufficient conductance. Such an ideal cylinder 100 A long would be 300 A in diameter,
assuming bulk conductivity.
The fusion of phospholipid vesicles to planar phospholipid membranes required
swelling of adherent vesicles. We found that anything we could do to swell an adherent
vesicle would lead to fusion, whereas we never saw fusion without osmotic swelling
(Cohen, Zimmerberg and Finkelstein, 1980; Akabas, Cohen and Finkelstein, 1984;
Finkelstein, Zimmerberg and Cohen, 1986). This requirement for swelling seems to be
absent in vesicle-vesicle fusion (Duzgunes, 1985; Duzgunes, Wilshut, Fraley and
Papahadj opoulos, 1981), but the lateral stress provided by swelling may be important.
Aggregation by itself will cause fusion in vesicle-vesicle interaction, because adhesion
will promote stress (Kachar, Fuller and Rand, 1986; Zimmerberg, 1987). So, in the
interaction of two topologically closed systems, whenever there is attraction and
adhesion there must also be deformation and a build up of internal pressure, but with
Molecular Mechanisms of Membrane Fusion
DC measurement
255
AC measurement
Vln
Vin
,
[
Cg
Zg= mCg
} Rv
,~.
C
Vin
•1•
Vin
" --Zg
Vin
I= R'v~+
Rp
d
Vin
Zg+Rp
m
Fig. 2. Detection of pore resistance. DC measurement: A planar membrane is formed between two
electrodes and a potential, Vin, is applied across that membrane. Vesicles containing channels are
added to one side of the planar membrane (cis side). When a pore forms between the vesicle interior
and the solution on the trans side of the planar membrane, current, I, passes through the series sum of
the vesicle resistance, Rv and the pore resistance, Rp. This current will be maximal for Rp ~ Rv. If Rp
is initially infinite (no fusion, Fig. la,b)) and becomes zero, (fusion, Fig. le), then current will be
initially zero and increase to Vin/Rv. If Rp is comparable to Rv (Fig. lc,d) for times longer than the
instrumental limitations, then current will gradually increase from zero to Vin/Rv. Rp can be
calculated from the series expression I=Vin/(Rv+Rp). Experiments to data show only
instantaneous changes in Rp (see text). AC measurement: A cell is voltage-clamped in the tight-seal
whole-cell configuration. An alternating potentisl, Vin, is applied between the inside and outside of the
cell. A capacitor offers resistance to alternating current. This a.c. resistance is termed impedance, Z.
The impedance is related to capacitance by the relationship Z = 1/coC. The impedance of a resistor is
equal to its resistance. The amplitude of the current is inversely proportional to the impedance. When
an exocytotic pore forms between a granule and the plasma membrane, current passes through the
granule impedance, Zg, and the pore impedance, Rp in series. As the pore is initially closed and its
resistance infinite, no current flows through the granule until Rp is comparable to Zg. During the
time of pore widening, Rp can be calculated from I = Vin/(Zg + Rp). By using a lock-in amplifier, the
capacitative and resistive elements of the current change during fusion can be dissected (Zimmerberg,
Curran, Cohen and Brodwick, 1986).
o n e o p e n , r e l a t i v e l y l o w s u r f a c e e n e r g y m e m b r a n e t h a t is n o t n e c e s s a r i l y t h e c a s e
( E v a n s a n d P a r s e g i a n , 1983). T h e p l a n a r m e m b r a n e has a large t o r u s of d e c a n e a n d
p l h o s p h o l i p i d f r o m w h i c h t o d r a w m a t e r i a l to a l l o w a n i n c r e a s e in surface a r e a at little
e n e r g e t i c cost. T h i s difference b e t w e e n t h e s e t w o s y s t e m s is i m p o r t a n t in d e s i g n i n g a n
a s s a y s y s t e m for t h e v a r i o u s f u s i o n p r o t e i n s , w h e r e o n e wishes to m e a s u r e f u s i o n
independently from adhesion.
Discharge
A n o t h e r m e t h o d o f d e t e c t i n g f u s i o n d e t e r m i n e s the t r a n s p o r t of v e s i c u l a r c o n t e n t s
a c r o s s t h e m e m b r a n e to t h e e x t r a c e l l u l a r space. W e effected this in vesicle to p l a n a r
256
Zimmerberg
membrane fusion by placing a fluorescent marker inside the vesicle and watching its
appearance on the side opposite the one to which we added vesicles (Zimmerberg,
Cohen and Finkelstein, 1980). Swelling adherent, dye-filled vesicles led to dye release
which appeared as a burst of light when the dye had been at a self-quenched
concentration (Woodbury, 1986c; Niles and Cohen, 1987b; Niles and Cohen, 1987a).
Adding quenching agents to only one side of the membrane allowed Niles and
Cohen to distinguish fusion from lysis (Niles and Cohen, 1987b; Niles and Cohen,
1987a). They saw about 50 % fusion upon swelling. Similar ratios have been calculated
for content mixing in vesicle-vesicle fusion but this is dependent upon lipid and salt
composition (Kachar, Fuller and Rand, 1986; Duzgunes, 1985; Walter, Steer and
Blumenthal, 1986).
BIOLOGICAL MEMBRANE FUSION
We have been recently using two systems for studying exocytosis: one, the egg of
the sea urchin discussed in detail in the article by Michael Whitaker on page 383 of this
volume; the other the mast cell of the beige mouse.
The beige mouse is an animal model for the Chediak-Higashi syndrome, an
immuno-suppressive disorder in humans which is associated with large secretory
granules in granulocytic leucocytes (Chi and Lagunoff, 1975; Lutzner, Lowrie and
Jordan, 1967). The mast cell of the beige mouse has a few, very large, secretory granules
(Fig. 3). We can trigger exocytosis directly by intracellular perfusion of the mast cell
with GTP-Gamma-S, ITP, and low calcium using the whole-cell patch pipette
technique (Neher and Marty, 1982; Fernadez, Neher and Gomperts, 1984). A patchclamped cell is shown in Fig. 2. The pipette is out of the focal plane, and the top of the
white border surrounding the oscilloscope gives the dimensional marker of 10 microns.
With this cell, we can study exocytosis physiologically and morphologically on the
single granule level.
Adhesion-Dehydration
This first step in fusion has not been systematically studied with biological
membranes. In the echinoderm egg and the mouse mast cell, the granules appear to be
pushed up against the plasmalemma, and physical disruption of the cell preserves
granule-membrane contact. The fact that these granules do not undergo brownian
motion indicates attachment, but there is no information about the proximity or
hydration of the attachment site. Using osmotic stress, Peter Rand and co-workers
measured the force-distance relationship of myelin, a multilayer of biological
membrane, and found the kind of repulsive hydration force described above (Rand,
Fuller and Lis, 1900).
Observation of exocytotic specimens with the electron microscope has revealed a
variety of putative pre-fusion structures (Chandler, 1987; Plattner, 1981). In the sea
urchin egg, we saw the attachment site as an electron dense region, using rapid-freezing
freeze-fracture, we found membrane separations of < 50 • (Zimmerberg, Sardet and
Molecular Mechanismsof MembraneFusion
257
Epel, 1985). In the mast cell, a separation of no less than 300-500 A, was seen
(Chandler and Heuser, 1980).
Molecular Rearrangement-Pore Formation
In biological exocytosis the melding of membranes can be measured by the
appearance of a new function (H § ATPase transport, ADH response) in the plasma
membrane, or membrane continuity as determined by increased surface area in
electron microscopy, or the appearance of omega figures in electron microscopy
following stimulation (Schwartz and A1-Awqati, 1986; Wade, 1986). Exocytosis can
also be measured through capacitance, the ability of a surface to store charge (Neher
and Marty, 1982). When a membrane is between two electrodes one can measure the
ability of that membrane to store charge. The constancy of specific capacitance,
~- 1 #F/cm 2, in a large variety of cell types suggests its use as a measure of cell surface
area. During exocytosis the cell capacitance increases due to the increase in cell surface
upon addition of vesicular membrane to the plasma membrane. The measured
capacitance will depend upon the access of current to the new membrane, so, as in
channel insertion during fusion of phospholipid vesicles to planar membranes, the full
capacitance will be seen when the pore has opened substantially (Zimmerberg, Curran,
Cohen and Brodwick, 1986).
Capacitance Flickers
Fernandez, Neher and Gomperts (1984) first demonstrated the presence of flickers
in capacitance: transient increases followed by decreases in capacitance. We also see
such events with beige mouse mast cells, e.g. the increase in capacitance, Fig. 3b,
followed by a decrease, Fig. 3c (Zimmerberg, Curran, Cohen and Brodwick, 1986).
During the flicker, capacitance often fluctuates noisily (Fig, 3d). Flickers are not
accompanied by any morphological changes, which may mean that there is a reversible
fusion which finally becomes stable. One consequence of flicker fusion may be nonquantal release.
The Exocytotic Pore in Biological Fusion
As in the model system, pore formation can be detected by the permeation of ions
between the secretory granule and the extracellular solution. Electrically, the ions
charging the secretory granule must pass through the exocytotic pore. This pore is in
series with the granule capacitance, and so the development of that pore can be
measured during exocytosis (Zimmerberg, Curran, Cohen and Brodwick, 1986). As
long as the exocytotic pore prevents maximal current across the granule membrane, its
resistance to current can be measured (Fig. 2c,d). By solving the equivalent circuit, we
have determined pore resistance during the rise-time of the capacitance measurement.
Exocytotic pore resistance often drops more slowly than the response-time of our
instrument (Fig. 3h). We have detected resistances between 2.5 • 109 and 3 x 108, or
conductances between 0.4 and 3 nS. If we assume bulk conductivity in the pore and a
cylinder 100 A long, we calculate pore diameters between 18 and 52 A.
258
Zimmerberg
Fig. 3. A sequence of events during exocytosis of mast cells from the beige mouse.
Sequential fields (17 ms) of one experiment are shown. Cells are internally perfused with
GTP-y-S and ITP with the whole-cell patch clamp technique (Marty and Neher, 1983).
Capacitance and conductance are measured in quadrature (Neher and Marry 1982).
Molecular Mechanisms of Membrane Fusion
Fig. 3 (contd.) Nomarski differential-contrast optics are used. The white border on top of
the oscilloscope inset is 10/~, Capacitance is shown in the top oscilloscope tracing (A.C.coupled, 50ms/sweep). The top digits in the middle directly below the cell display
conductance; capacitance is directly below. 100 units = 200 femptofarads or 1 nanosiemen.
259
260
Zimmerberg
Fig. 3 (contd.) Time in seconds and hundredths of seconds are shown at the b o t t o m
right. Flicker events: Although no morphological changes are seen in a-e, changes in
capacitance can be seen in the oscilloscope traces (arrows) and the digitized values (black
arrows). This activity soon ceased and capacitance stabilized at ~ 470 for the next 1.6
Molecular Mechanisms of Membrane Fusion
Fig. 3 (contd.) seconds (not shown). Fusion: The final fusion event is seen in e (arrow)
which continues in f. The capacitance increases instantaneously at first, then more slowly
to the peak. Swelling is seen first in f. The discharge is complete by g. In h another
capacitance change is seen, corresponding to the subsequent swelling of the top-most
granule.
261
262
Zimmerberg
Discharge
In biological exocytosis, content discharge can be measured morphologically,
chemically, o r enzymatically. Release is one of the unambiguous measures of
exocytosis, but may fail to occur despite membrane fusion (Whitaker and Zimmerberg,
1987). In cases where the discharge occurs faster than can be explained by diffusion,
one wonders whether there is a biological role for the secretory granule swelling
associated with exocytosis. The requirement for an osmotic gradient in the model
system prompts the question of a requirement for osmotic swelling in the biological
system. Shrinking a variety of cells in hyperosmotic solutions inhibits fusion
(Finkelstein, Zimmerberg and Cohen, 1986). One worry in this class of experiments is
that altering the tonicity of the external medium with cell shrinkage, may dislodge
granules from attachment sites, alter the cytoplasmic viscosity, change cytoplasmiccytoskeletal relationships, create more membrane folding, and concentrate
impermeant species. In systems in vitro, we also found an inhibition of fusion with
hypcrosmotic solutions (Zimmerberg, Sardet and Epel, 1985; Zimmerberg and
Whitaker, 1985). Holz and Senter, in their work on the chromaffin cell, came to the
conclusion that flaccid vesicles fused (Holz and Senter, 1986; Holz, 1986). Although
many results are consistent with the theory that swelling caused fusion, there is still the
nagging possibility that swelling was not causing fusion, and changing osmotic
pressure was altering the hydration state of some macromolecule that was crucial for
the fusion process itself. That swelling follows fusion had been proposed by others
(Green, 1978; Green, 1982; Schuel, 1978). However, transient swelling during fusion
cannot be ruled out in inhibition experiments (Whitaker and Zimmerberg, 1987).
The direct experiment for the osmotic hypothesis measures swelling and
membrane fusion simultaneously. If swelling precedes fusion, it may be driving fusion.
If swelling occurs after fusion, it can not be the driving force for membrane fusion. To
distinguish the sequence of events we monitored membrane fusion by capacitance
changes while simultaneously watching swelling with high-resolution differentialinterference-contrast video microscopy (Zimmerberg, Curran, Cohen and Brodwick,
1986).
A typical experiment is seen in Fig. 3. The resolution of the change in capacitance
is determined from the amplitude of the capacitance signal noise. This amplitude
ranged from 25 to 100 femptofarads depending upon the degree of filtering. The time
resolution is seen in Fig. 3e, in the capacitance risetime in the oscilloscope tracing. This
was as low as 2.3 ms. The morphological events were measured with a time-resolution
of 17 ms and a spatial resolution of 0.18 #, with a detection of swelling of 0.084 #. In all
experiments, the capacitance change (Fig. 3e) preceded granule swelling (Fig. 3f,g).
Again, swelling followed the fusion seen in Fig. 3h (not shown). Independently,
Breckenridge and Almers (1987) have confirmed these findings.
Were the granules already so taut that an indetectable swelling led to fusion? To
test this possibility, we performed experiments upon cells with hyperosmotic solutions
where we shrank the granules, then disrupted the cell membrane and placed granules in
isotonic medium (Zimmerberg, Curran, Cohen and Brodwick, 1986). We could visibly
swell these vesicles by ~ 12% before they would burst. Repeating the simultaneous
optical and electrical experiments with hypertonically treated cells also led to the
conclusion that membrane fusion preceded swelling (Fig. 4). Thus swelling of the mast
Molecular Mechanisms of Membrane Fusion
263
ce].l granule was not required for the fusion of mast cell secretory granule to plasma
membrane.
Living systems exhibit, at least in this system, some way of overcoming the barrier
to fusion seen in phospholipid membrane fusion. Is there a paradox between these
experiments and those in other biological systems suggesting that swelling might be
important for fusion? It depends on whether you are a monotheist or not. There could
be different mechanisms operating in other biological systems. When I told Peter
Baker about this result, he suggested that perhaps there is a role for swelling in terms of
stabilizing the final fusion; that if an opportunity at the beginning of fusion is not
completely realized, then a window of opportunity is lost and fusion is inhibited.
Hyperosmotic solutions might have a direct effect on the conformation of an essential
macromolecule, or on the hydrated pore which opens as an intermediate before the
detectable exocytotic pore.
MECHANISMS OF BIOLOGICAL FUSION
Kinetics of Pore Formation
In contrast to the immediate full fusion and large pore seen when phospholipid
vesicles fuse to planar phospholipid membranes, we see much smaller pore
conductances that are stable for milliseconds. Instead of a pore 300 A wide after less
than 50 gs, in the beige mouse we detect smaller pores widening over a range of times,
from faster than our ability to resolve, ~ 3 ms (Fig. 3e), to 100 ms (Fig. 4b,c). This is
very slow compared to lipid bilayer fusion. The difference in dynamics for the fusion
event may relate to the size of vesicles or be due to protein effects on surface rigidity.
Other exocytotic systems are presumably much faster, e.g. < 200 #s in synaptic vesicle
fusion in squid giant synapse (Llinas, Steinberg and Walton, t982). It is important to
stress that there is no information in the beige mouse system about how much time
elapses between the biological trigger for exocytosis and the first detectable pore. We
can only detect the pore conductance over a narrow range of conductances, during the
time when the impedance of the exocytotic pore is comparable to that of the granule. In
pictorial terms, this is the time for a transition from Fig. lc to ld. In fact, the
capacitance measurements are carried out in the frequency domain with oscillating
fields and analysis of capacitance and conductance is done with a lock-in amplifier. The
shape of the wave forms that we see during the actual change in capacitance is
consistent with the solved equivalent circuit of the series resistor and capacitor
(Zimmerberg, Curran, Cohen and Brodwick, 1986).
Major differences between biological and model membrane fusion are control and
speed. While fusion of phospholipid vesicles is accompanied by a certain degree of lysis,
biological membrane fusion is presumably not accompanied by intracellular lysis.
Certainly not in the pancreas, where intracellular spillage of protease during exocytotic
secretion would be disastrous. Such control is usually the domain of proteins. The fact
that exocytotic pores of small diameter are stable over many milliseconds puts
constraints on the type of hydrated surfaces which might make up such a pore
(LeNeveu, Rand and Parsegian, 1976; Rau, Lee and Parsegian, 1984). The putative
pore dimensions are remarkably similar to those found in proteins which form pores in
264
Zimmerberg
Fig. 4. Fusion of flaccid vesicles precedes swelling. Key as in Fig, 3. Cells were
treated in hyperosmotic medium prior to sealing. The fusion event is seen in b (arrow) as
Molecular Mechanisms of Membrane Fusion
Fig. 4 (contd.) an increase in capacitance. Swelling is not seen until after c, and is evident
in d.
265
266
Zimmerberg
single phospholipid bilayers (Zimmerberg and Parsegian). The role of hydration in
such spaces is currently under investigation. While certain lipids will form cylinders
composed of aqueous cavities approximately 20 ~ in diameter, this is with lipids which
are not happy in the lamellar phase (Gruner, Parsegian and Rand, 1986). The
formation of a gap-junction like structure between secretory granule and plasma
membrane may, then, be composed of either enzymatically altered lipid or protein
(Finkelstein, Zimmerberg and Cohen, 1986). One guess is that a multi-subunit protein,
a barrel former, might hydrate upon contact leading to membrane fusion.
Thus membrane fusion, a biological phenomena, can be induced in phospholipid
bilayer membranes. The driving force for fusion in that system cannot drive the fusion
of beige mouse mast cell granules during exocytosis. Two major avenues of research
are apparent. First, one could make further observations of the sequence of events in
other exocytotic systems, and in more detail in the mast cell. The exocytotic pore may
be defined by additional probes, such as fluorescence. Stabilization of the pore would
greatly aid study of the flicker phenomena. Secondly, one could try to produce fusion
phenomena more similar to biological fusion. Phospholipid bilayers can be fortified
with addition of putative fusion proteins isolated from biological material. Particularly
promising are proteins isolated from temperature-sensitive mutants in secretion in
yeast or mating mutants in yeast and Clamydomonas. Viral fusion proteins are also
available with specified alterations and some degree of defined structure.
Reconstitution with defined constituents will provide new opportunities to learn the
physical forces and structural modifications of macromolecules responsible for
membrane fusion.
ACKNOWLEDGEMENTS
This paper is dedicated to the memory of Peter Baker. I first saw Peter Baker in
1978 at a conference on calcium in New York. It seemed that he was the only person
who could integrate and explain all of the isolated findings on intracellular calcium
regulation. His clarity was most inspiring. In other meetings and conversations on
membrane fusion and exocytosis, his remarkable ability to bring the discussion quickly
to crucial yet rarely discussed questions provided a rare source of intellectual
communion. I will miss him very much.
REFERENCES
Akabas, M. H., Cohen, F. S. and Fmkelstein,A. (1984).Separation of the osmoticallydriven fusion event
fromvesicle-planarmembraneattachmentin a modelsystemfor exocytosis.J. Ceil. Biol. 98:1063-1071.
Breckenridge,L. J. and Almers, W. (1987).Final steps in exocytosisobservedin a cell with giant secretory
granules. Proc. Nat. Acad. Sci. 84:1945-1949.
Boys, C. V. (1959).Soap Bubbles. Dover Publications,Inc., New York, NY, pp. 101-103.
Chandler, D. E. (1987).Current topicsin membranesand transport, exocytosisand endocytosis:membrane
fusion events captured in rapidly frozen cells. Dev. Biol. 5:435-459.
Chandler, D. E. and Heuser,J. E. (1980).Arrest of membranefusioneventsin mast cellsby quick freezing.J.
Cell Biol. 86:666-674.
Chi, E. Y. and Lagunoff, D. (1975). Abnormal mast cell granules in the beige mouse. J. Histochem.
Cytochem. 23:117-122.
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