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Cytomimetic Organic Chemistry Early Developments.

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REVIEWS
Cytomimetic Organic Chemistry: Early Developments
Fredric M. Menger* and Kurt D. Gabrielson
~~~~
This article describes how chemical and
physical stimuli cause a simple system,
the giant vesicle, to undergo a variety of
“cytomimetic” transformations such as
fusion. fission, endocytosis, budding,
aggregation. birthing, and foraging. For
example. when a giant vesicle, which
happens t o have a smaller vesicle inside
it. is exposed to octyl glucoside. the
smaller vesicle can pass through the outer membrane into the external medium
(“birthing”). The resulting injury to the
membrane of the host vesicle heals immediately. Addition of cholic acid, on
the other hand, induces a feeding frenzy
in which a vesicle grows rapidly as it
consumes its smaller neighbors. After
the food is gone, the giant vesicle then
self-destructs (a case of “birth, growth,
and death”). Such lifelike morphological changes were obtained by using coinmercially available chemicals; thus these
1. Introduction
The ancient Greeks believed that conversion of an unorganized universe (“chaos”) into an organized one (the “cosmos”)
was the work of their dieties. Today, organization (especially in
complex biological systems such as the cell) seems almost as
puzzling as it did centuries ago. I n mysterious ways, molecules
organize themselves into elaborate assemblies to produce a phenomenon called “life”. Since living systems presumably obey
the fitndamenial laws of chemistry, biological behavior must, by
default. originate from its collective and holistic nature. Biology
is, in eff‘ecl, organized organic chemistry. The question then
becomcs: Are the properties of organized organic molecules
predictable from the familiar properties of individual molecules’! O r in other words: Are organic “systems” simple extrapolations of single-molecule chemistry? We have already argued
negatively t o these questions.[”To learn about organized assemblies. and ultimately about biology, one must forsake singlemolecule organic chemistry and confront the assemblies themselves despite the difficulties, complications, and uncertainties
that will undoubtedly ensue.
Consider the role of the rr-meson in chemistry. Since xmesons are components of all nuclei including that of carbon,
organic chemistry necessarily embodies them. A decarboxylation, for example, entails a loss of rr-mesons. Why then are
chemists completely indifferent to these elementary particles?
[*] Prnf I- hl Mcnger. Dr. K D. Gabrielson
I)cp;iiIincnr oI‘(’hem~str). Emory Uni\crsity
Allanta. O A 10327 (USA)
Telel,ix’ 1111 c o d c
(404)727-65X6
+
processes should be assigned to organic
chemistry, and not to biology or even
biochemistry. Experimental details (e.g.
the preparation and observation of the
vesicles) are included in this review in
hopes of helping others enter this undeveloped field.
Keywords: cytomimetic organic chemistry * microscopy - supramolecular
chemistry vesicles
The answer is that chemists operate at a level of complexity and
organization where particle physics is of absolutely no consequence. Three related quotes reinforce this general point. W. H.
Thorpe: “The behavior of large and complex aggregates of elementary particles is not to be understood as a simple extrapolation of the properties of a few particles. Rather. at each level of
complexity entirely new properties appear”. P. W Anderson:
“At each level of organization, types of behavior open up which
are entirely new and basically unpredictable from a. . .detailed
analysis of the entities which make up the.. .higher level systems.” And A. R. Peacocke: “Higher levels of complexity are
distinguished by some genuinely new features and activities, and
these require distinctive theories, language, and concepts to describe them”. I t is the philosophy embraced in these quotes that
motivated the work that will be described in the following pages.
Vesicles, also called liposomes, are spherical structures composed of a closed lipid shell surrounding an aqueous interior
(Fig. 1). Under certain conditions, lipids can self-assemble into
giant vesicles the size of living
As will be detailed later,
giant vesicles engage in many cell-like activities such as aggregation, budding, fusion, and fission.[31We refer to this behavior
as “cytomimetic” chemistry.[41
Although we are hesitant to introduce such new jargon, the
descriptor cytomimetic for the
striking morphological changes
H2
7.“
(visible under a light micro-
J. S. Mill, “Hardly any thoughts
CUICF
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ever make their way among mankind. even in the minds of their
inventors, until aptly selected words nail them down and hold
them fast”.
We are hardly the first to become fascinated by cytomimetic
chemistry. Over sixty years ago, in a brilliant piece of work,
Crile et al.[’] combined the ether-soluble fraction of brain tissue
(i.e. the lipids) with the water-soluble fraction (i.e. the proteins
and nucleic acids) .161 Within minutes remarkable structures,
called “autosynthetic cells”, formed in the water. Each contained a microscopically visible central organelle resembling a
nucleus. The structures grew slowly, multiplied by budding o r
direct division, and occasionally moved like an amoeba. They
even exhibited a high rate of oxygen consumption. Although the
findings have been confirmed independently,[’, ‘1 they have been
largely ignored. We are currently resurrecting this work (the
desire to d o so being irresistible!), but the present article will
deal only with much simpler systems composed of material that
is not of direct biological origin.
It is necessary to stress the preliminary nature ofwhat is about
to unfold. Work began just two years ago in a small office made
over to house a microscope. The plan was to watch the response
of giant vesicles as they were exposed to chemicals, heat, touch,
and various other irritations. At this early stage of research, we
hoped more for a slow accumulation of information than for
instant illuminative insights. J. F. Wilford described the situation
succinctly: “Science proceeds first by open exploration, the time
of initial discoveries. Then follows reconnaissance, gathering
evidence on a broad front, pursuing leads to the far corners of
the problem, and generating preliminary hypotheses. Finally, if
warranted by the results of the exploration and reconnaissance,
there comes the time for detailed studies and the testing of more
informed hypotheses”. By all counts, we are in the “open exploration” phase. Yet even so, the results to date have been sufficiently intriguing that their publication, in hopes that others join
the enterprise, appears justified.
Since this article is intended for a broad range of nonexperts,
an attempt has been made to avoid a technical, dehydrated style
of writing. This was easy to d o because “when we begin the
study of any science. we are in a situation, respecting that science, similar to that of children” (A. Lavoisier). Indeed, many
were the times when we gazed with a child’s wonder at the
dynamics of our giant vesicles.
i
i
i
~
1
F. M. Menger and K . D. Gabrielson
-
. -
2. A Brief History of Giant Vesicles
A. Einstein said “A hundred times every day. I remind myself
that my inner and outer life are based on the labors of other
men, living and dead, and that I must exert myself to give in the
same measure as I have received.” We too owe a debt to others
who, before us. investigated giant vesicles and related systems.
In 1911 Otto Lehmann wrote a book entitled “Die Fliissigen
Kristalle” (“Fluid Crystals”) in which he published optical
micrographs of long tubules projecting from the edges of hydrated phospholipid films.f21He also observed vesicle structures
that he called “kunstliche Zellen” (“artificial cells”). As we shall
see. Lehmann’s tubules were important to our own research
carried out eight decades later. D. Hammarskjold once asked.
“Why this desire in all of us that, after we have disappeared, the
thoughts of the living shall now and again dwell upon our
name?” Otto Lehmann certainly had, at least in our laboratory,
his wish fulfilled.
For all practical purposes, Lehmann’s system lay dormant
until the mid-1960s when it was revitalized by Alec Bangham.
(Vesicles, in fact. were once referred to as “bangosomes”).
Bangham was among the first to appreciate fully what now
seems to be a rather obvious fact: Vesicles separate an interior
volume from the external solvent by means of a semipermeable
barrier (i.e. the lipid bilayer) .Ig1Substances placed within vesicles can, therefore, be protected (at least temporarily) from the
surrounding bulk medium. This, plus the ability to target vesicles to specific biological sites, has stimulated considerable commercial interest. Since prospective uses of vesicles in pharmacology, medicine, cosmetics, diagnostics etc. are discussed
elsewhere.[”] little more will be written about them here. Suffice
it to state that the mundane lipid is a particularly interesting and
useful substance not because of its molecular structure per se but
because of its aggregated form. Organized lipid assemblies are,
accordingly, ubiguitous in Nature. Even “our souls may reside
in fat” (E. Chargaff).
The bulk of vesicle research has employed so-called small
unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs).[’ll Both are submicroscopic structures (about 30-50 nm
and 100-200 nm in diameter, respectively). A typical SUV
preparation is carried out as follows:[’21Lecithin or some other
phospholipid (20 mg in 200 pL CHCI,) is placed in a 4-dram vial
Fvedric M. Menger was horn, ruised. and educuted in the Northern
temperate zone. His,fi,ndnrss,fiv quotes. eltdent in the test, prompts
one ,from the late rock slur Frunk Zuppa: “I w i f e hecuusc I urn
personal(^^ amused at what I do”. Realizing that his projkssorial desk
is ri dangerous I3hce ,from which t o view the ~ ( i r l c lhr
, trtivels esfensivelj>to ,fav;flun,o places, qfirn carr.)ing his hlues hrimionicm with
Kurt D. Gabrielson carried out most qf ihe experinienis described in
this article. He hus ci BS,fiom Rhode Island Collegp, and ha recentlj~
rrceived his Ph.D. ,front Em0r.y. He is no,z’ working,fi?r the Grorgiu
Pacific Corporation.
’
F M . Mengci
K D. Cabrielnon
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Cytominietic Organic Chemistry
and the solbcnt evaporated with a stream of nitrogen. The resulting lipid film is then dried under reduced pressure, mixed
with 2.0 mL buffer, and sonicated (probe or bath) for up to 15
min at a temperature exceeding the phase-transition temperature of the lipid (70 .C being sufficient for most lipids). Dynamic
light scattering can be used to determine the size distribution of
the resulting SUVs. LUVs are commonly prepared by rapidly
injecting an alcoholic solution of the lipid into buffer."31 Alternatively. an ether solution of the lipid is slowly injected into a
warm aqueous phase where the ether evaporates, and the lipid
assembles into large vesicles.[I4'
Thrce disadvantages of SUV and LUV systems are worthy of
note. a ) Generally, SUVs and LUVs display a rather broad size
distribution. This can present a problem if the various sizes
within a population d o not have identical properties (pernieability, spectra etc.). b) SUVs and LUVs, being of small diameter,
possess a high curvature compared to the much flatter cellular
membranes. Curvature perturbs lipid packing and affects, therefore, the membrane behavior. c) Since one cannot visualize SUVs and LUVs under a light microscope, much information about
them is necessarily difficult to obtain. It is not easy, for example,
to differentiate SUV fusion from SUV aggregation. Undulation
of membrane bilayers is almost impossible to monitor. And
SUV;LUV systems d o not lend themselves to the studies of
injury and hcaling processes within the membrane. For all these
reasons. we decided to switch to so-called giant vesicles that can
be directly visualized under the light microscope.
At one time. when we were deeply immersed in SUViLUV
r e ~ e a r c h . l ' ~we
' invoked the three famous questions of Kant:
What can I know? What ought I d o ? What may I hope? With
regard to thc first question, we felt that there were important
processes (e.g. membrane injury and healing as mentioned
above) that could t i o f be properly defined by using SUViLUV
systems. Therefore. in response to the question of what ought to
be done, we thought it advisable to shift to giant vesicles. What
may be hoped by doing this? That. of course. is a main theme
o f this paper.
Before proceeding, it might be useful to discuss an important
parameter: vesicle size. Table 1 gives, in various units. the diameters of different types of vesicles and biological objects.
Tiibls I.
I ~ i . i i n c ~of
~n
v ~ i - i o chemical
u~
iniccllc
suv
LIJV
r'ihies v i r t i \
lyphvld bacillu\
red hlood cell
.imoch;i
giant \.cs1cIc
intended to be, in part, a primer on giant vesicles, it is worthwhile to summarize the methods that have been reported for
their preparation.
2.1. Drying-Rehydration Method[161
Phospholipid (1 - 3 mg) is dissolved in 1 mL CHCIJMeOH
(1O:l) in a 5 mL-round-bottom flask. The solvent is then removed by rotary evaporation (keeping the flask a s horizontal as
possible to maximize the film area). About 5 mL deionized water (or aqueous solution with less than 1 mM salt) is added carefully so as not to disturb the lipid film, and the flask is placed in
warm water (70 'C) for 1 hour during which the hydrated film
separates from the walls of the flask (Fig. 2 ) . On continued
1=:
24/40
Fig. 2. The drying-rehydration method for making _elan( iericle\. Phospholipids
are often hydrated at 70 'C, whereas 50 C is sufficient for did~)dccqIdimettiylammonium bromide (DDAB).
and biological entities.
x
nm
50
300-500
1000-2000
I300
7000
1 x lo*
l o x 10'
11.5 2iJn10'
5
30- 50
100 - 100
130
700
7 x 103
10 x lo*
0 . 5 - 1 0 x 10'
Pin
0.1 -0.2
0 .I 3
0 70
7
I00
5-200
inm
0.007
0.10
0.01 0.20
Since giant vesicles equal or exceed cellular dimensions, they are
microscopically visible. Although giant vesicle preparations are
usually polqdisperse (multisized), this takes on far less significance cornpared to SUV/LUV systems because. as will be
shown, we select and operate on only one giant vesicle at a time.
And the size of this vesicle is always known. Since this article is
heating (4 hours to 2 days), the lipid forms ;I single globule,
perhaps 1 cm in diameter, that resembles a translucent sea-creature suspended in water. The flask is removed from the bath and
shaken for only a few seconds (no more!) while it is still warm.
This causes the globule to disintegrate into a polydisperse collection of giant vesicles suspended in a nearly clear solution. The
vesicle size distribution depends upon the incubation time as
well as the duration and vigor of the shaking step. Modifications
of this procedure are described in the literature." 'I
2.2. Dialysis Method["]
In this approach, 1-0-methylglucoside (200 mg) plus lipid
(10 mg) in 400 pL methanol are dialyzed against pH =7.4
buffer for two days. Giant vesicles are formed under conditions
'093
REVIEWS
where their internal contents are continuously hyperosmotic.
Diameters range from 10- 100 pm. It is claimed that the vesicles
are mainly unilamellar (a single bilayer) as opposed to multilamellar (several bilayers in an onionlike configuration).
Sodium trichloroacetate can also be used to achieve an osmotic
gradient across the bilayer.[’”] The dialysis method has an advantage over drying-rehydration in that it can be used with high
salt concentrations. On the other hand, possible contamination
by the “indifferent solute” is an ever-present concern.
2.3. Freeze-Thaw Method[’’]
Giant vesicles are produced from SUV systems. generated in
water by sonication. when the latter are repeatedly frozen in
liquid nitrogen and then thawed. This method is advertized as
being particularly suitable for lipid systems containing proteins
because drying-rehydration and dialysis could conceivably denature the proteins.
2.4. Solid Hydration
From our point of view, it was especially important to minimize formation of rnultilamellar (onionlike) vesicles owing to
their complexity. Vesicle fusion, for example, becomes difficult
to interpret when multiple layers of varying curvature collectively get into the act. In our hands, all three of the above methods
(especially dialysis and freeze-thaw) gave significant populations of multilamellar vesicles with both phospholipids and synthetic surfactants. We discovered, however, a fourth method
that gives >90°/0 unilamellar vesi-
\ N,,cH3
cles with a synthetic lipid, didodecyldimethylammonium
bromide
Br(DDAB).[41
/ \
C H ~ C H ~ I I CH3
In this method, DDAB (0.1DDAB
0.2 mg) was placed within a rubber
washer (14 mm i.d.) cemented onto
a glass slide. The powder was then immersed i n 450 pL water
and allowed to hydrate. (The amounts of DDAB and water are
important; too much DDAB gives multilamellar vesicles: too
little DDAB results in only a few vesicles by light microscopy).
Hydration begins instantly a s seen by wormlike tubules (myelin
figures) forming at the surface of the solid particles (Fig. 3 ) .
After about five minutes, one observes many grapelike clusters
some of which transform into 5-10 pm vesicles. Remaining
CH3(CH2) I
Fig. 3 Hydration of powdered D D A B as ohservcd by phase-contrast microscopy.
A . Initial formation of tubules. R . Subsequent formation of‘giant unilninellnr vesicles (GUVs).
2094
F. M . Menger and K. D. Gabrielson
clusters develop (via a poorly understood fusion process requiring about two hours) into giant unilamellar vesicles. These were
easily distinguishable, by phase-contrast microscopy. from the
small population of the more opaque multilamellar vesicles.
Giant unilamellar vesicles will henceforth be symbolized by
GUVs.
The preceding recipe works well for DDAB but not so well for
phospholipids. In our experience, the conditions for making
GUVs must be worked out individually for each particular lipid
and temperature. Since DDAB gave such excellent preparations
(even in the presence of additives such as cholesterol), and since
the preparations displayed such a variety of cell-like morphological changes, we used DDAB exclusively for our initial studies. There is no reason in principle, however, why our experimental approach cannot be extended to all sorts of lipids,
natural and synthetic alike.
Giant unilamellar vesicles, prepared by hydrating solid-state
DDAB, are stable for two-three days after which they tend to
aggregate. Note that GUVs d o not lie in a global energy minimum despite their seemingly spontaneous formation.[211The
global energy minimum is in fact occupied by flat bilayer sheets;
energy input is required to impose curvature upon such sheets.
Convection currents and vibrations (usually unnoticed) suffice
to achieve the mild curvature in GUVs (while, as mentioned,
sonication generates high-curvature SUVs). Since GUVs,
tubules, and many of their structural variations are all relatively
flat, and thus of similar energy. a rich array of interconvertible
morphology is possible.
Before continuing, the reader can rightly expect answers to
some basic questions. the most important of which is: How d o
we know that our GUVs are indeed unilamellar (i.e. endowed
with only a single lipid bilayer)? This is far from a trivial question as evidenced by the fact that we initially mistook multilamellar vesicles for unilamellar structures. The confusion arose
from three sources: a) Literature reports on giant vesicles assume, often without sufficient supporting data, a unilamellar
assembly. Initially, we did likewise. b) As mentioned above,
unilamellar vesicles are far more desirable than multilamellar
vesicles. We should have kept in mind the quote of H. J. Van Till:
“The purpose of empirical research is to discover what the physical world is really like, not to verify its conformity to our preferences”. c) Finally. a s chemists we were novices with the light
microscope. The unilamellar issue could not be resolved until we
had become experienced in its use.
Vesicles turned out to be invisible by ordinary bright field
microscopy because the refractive indices of the vesicle and
aqueous medium are too similar. We thus called upon phasecontrast microscopy which is particularly useful in such cases.
In phase-contrast microscopy. a hollow cone of light passes
through the sample, and in this manner one achieves a variation
in brightness across boundaries even when the difference between refractive indices is small. Phase-contrast illumination is
easily produced with an inexpensive metal ring that is slipped
into the microscope’s condenser. Further details of phase-contrast optics can be found elsewhere.[221
Figure 4 shows phase-contrast photomicrographs of giant
DDAB vesicles (ca. 90 pm in diameter at 400 x magnification).
Although the vesicles are spherical, they appear flat because the
microscope’s depth of field (1.5 pm with a 40 x objective lens)
REVIEWS
Cytomimetic Organic Chemistry
Fig.
I'hav-corilr~ist pholornicrographs ol'a multilemellnr giant reside (A).
ii unil;rmellnr giant vesicle ( D )
a11
~ d i g ~ ~ l ~ i i ipi;int
~ c l l :wsiclc
~r
( B m d C), :ind
is much less than the vesicle diameter. Phase-contrast microscopy can "see" only a narrow slice of lipid at the equator;
attempts to focus above or below the equator produce only a
blur. A typical multilamellar vesicle is given in Figure 4A. I t is
readily identified as such by an opacity and texture that differ
from the medium surrounding the vesicle. The vesicles in Figures 4B and 4C are also multilamellar. but the layers can actually be resolved because they are, in this case, less densely packed.
Finally. Figure 4D shows a giant unilamellar vesicle, the type
that is most desirable for membrane research.
A lipid bilayer, being only about 40 8, thick. is obviously
impossible to see by light microscopy. Thus. the dark ring in
Figure 4D is an optical effect; in no way does it portray the
actual membrane thickness. Similarly, light microscopy cannot
differentiate between a membrane composed of a single bilayer
(a true GUV) and a membrane composed of a few bilayers.
Although the distinction makes little difference from the point
of view of our experiments, we believe that true unilamellar
vesicles are indeed formed for the following reasons: a) When
GUVs are chemically o r physically destroyed, the ring disapunit, leaving no visible inner ring. (In contrast. multilamellar vesicles are often seen to "peel o f f ' their layers one by
one). b ) Published cryoelectron micrographs on vesicles similar
to those in Figure 4D suggest the presence of a single-layered
c o n t i ~ u r a t i o n . l 'c)
~ ~So-called bilayer lipid membranes (BLMs),
in which ii 60-100 8, film spans a 1 mm hole in a Teflon
prove that one o r two bilayers are capable of maintaining struc[ural integrity over macroscopic distances.
Among the rather limited literature on giant vesicles, ii few
articles stand out as having been particularly relevant to our
work. These will now be reviewed briefly.
Mueller et a1.[2s1published early experiments on giant vesicles
prepared (by the dehydration-rehydration method) from brain
lipids, egg lechithin, and synthetic phospholipids. A number of
points are of interest: a) The vesicles can be made in distilled
water and in solutions of non-electrolytes but not in salt solutions where the lipids precipitate. b) After growing in size for
several days, the vesicles are stable for weeks. c ) Vesicles can be
fractionated according to size by centrifugation in specially designed tubes. Apparently, the giant vesicles can survive 40000 g
for 30 min (our DDAB vesicles being too fragilc to tolerate such
abuse). d ) Vesicles made in distilled water seem to be under
osmotic stress (of unknown origin) because physically rupturing
them causes interior contents to be rapidly expelled. e) Finally,
and this is important, the micrographs of Mueller et al. were
obtained by dark-field microscopy. This technique (which, like
phase-contrast, uses a special attachment in the condenser) portrays the vesicles as a ring of light in an otherwise dark field
(Fig. 5). In our experience,
such a ring of light is seen primarily with multilamellar
vesicles. One should resist the
temptation to assume that a
single shell in dark-field microscopy necessarily signifies
a unilamellar structure.
Zimmerman et al.["I observed by light microscopy an
electric-field-induced fusion
Fig 5, Dark.,icld photoinlcrograpl,
of giant vesicles prepared
o f giant m i ~ ~ t i i a r n e ~vesicles.
~ar
from a 1 : l mixture of a
cationic lipid and cholesterol. Application of an alternating electric field (200 Vcm- 100 kHz) first caused the vesicles to move
into direct contact with each other (Fig. 6). The fusion process,
requiring about one second, was initiated by turning off the AC
field and subjecting the sample to a 3-9 kVcn7-I field pulse of
20-50 ps duration. The electric breakdown o f the membrane
creates, it is postulated. large numbers of aligned pores in the
contact zone (Fig. 6). Reorganization of lipid then bridges the
n
t3
0
........
3. The Literature
When the plan of studying giant vesicles was first initiated, we
knew littlc about how to prepare, observe, and manipulate
them. "Between the idea and reality stands the shadow" (T. S.
Elliot). Past work of others helped illuminate the "shadow".
Fig. 6. Fusion induced by
iiii
clectric lield as adiiptcd li-om rcf 1261
2095
F. M . Menger and K. D. Gabrielson
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1
COOH
H d
AcHN
i
‘
1
nated to a crescent. This extreme sensitivity of morphology to
two giant vesicles while simultaneously creating microscopically
temperature was not observed in our experiments with the relatinvisible SUVs that become trapped within the new vesicle.
The research group of H. R i n g s d ~ r f [ ”injected
~
a polysacchaed DDAB vesicles. Membrane morphology obviously depends
upon lipid structure in ways that are not yet understood.
ride, isolated from E. coli and called K I , into a giant vesicle
E.xperiment 2:1291Giant vesicles were prepared from a 4 : l
preparation. I t was found that K1 inserts itself into the vesicle
mixture of dimyristoylphosphatidylcholine (DMPC) and a
bilayer (presumably by means of Kl’s lipid moiety). Proof for
polymerizable phospholipid 2. When polymerization was
binding of K1 to the vesicle was obtained by adding a fluoresinduced by UV irradiation, the vesicles were seen to invaginate
cent-labeled anti-K 1 antibody to the preparation. Fluorescent
microscopy showed giant vesicles
with brightly fluorescent peripheries,
CH~(CH~)I~COO-CH~
I
clearly indicating that the antibody
CHdCHz)izCOO-~H 0
had concentrated itself at the exter+
I
ll
I
1
I1
nal membrane surface. In the abCH20PO(CH2CH20)40CO-C=CH2
CH?OPOCHZCH~N(CH~)~
I
sence of K1, the antibody fluores- 1
I
0
ONa’
cence remained distributed uniDMPC
L
formly throughout the aqueous
medium.
(a photochemically-induced endocytosis, Fig. 8). The explanaIt should be mentioned that the above work is only one of
tion given for the phenomenon rests on two assumptions: a)
several beautiful experiments described in reference [27]. The
Polymerizable lipid is of slightly higher concentration in the
1988 review (which also discusses endocytosis. hole formation,
outer leaflet of the membrane bilayer than in the inner leaflet
vesicle domains, and polymerization processes) is “must” reading for anyone entering the field of giant vesicles. We ourselves
owe it a special debt.
Among the diverse membrane papers of E. Sackmann. one
finds a few devoted to the giant vesicle. Four experiments in
particular will be mentioned here:
E,xperimmt 1 :[281 Giant vesicles were prepared from the quaternary ammonium lipid I, which has a phase-transition temFig. 8. A photochemically induced “endocytosls” with a giant vesicle composed of
a 4: 1 mixture of dimyristoyphosphatidylcholine (DMPC) and a polymerizable
perature of 42 “C.(A phase-transition temperature is the point at
phospholipid [29].
CH3(CH2)1,CH=CH-CH=CH-COO(CH
Br
/N\
CH3(CH2)j,CH=CH-CH=CH-COO(CH2)2
CH?
1
which a lipid transforms from a highly ordered gel state to a
more disorganized liquid crystalline state). The normally spherical vesicle took on a discoid shape at 42 “C (Fig. 7). When the
temperature was raised to 42.5”C, the form of the vesicle
changed to a “bowling pin”, while at 41.5 ”C the vesicle invagi-
41.5%
42’C
42.5%
Fig. 7. Shape transformations in ii giant vesicle composed of a synthetic lipid as the
temperature is raised and lowered 0.5 ‘C above and beiow the phase-transition
temperature of 42 C (281
2096
(the former possessing slightly more room for the bulky headgroup). b) Polymerization decreases the area per molecule. If
both factors are operative. polymerization will shrink the outer
leaflet more than the inner one, and the composite bikayer will,
therefore, distort inwardly. Svetina and Z e k ~ , ~ ~in’ ]a classic
article, were the first to propose that alterations in bilayer curvature arise because the two-component leaflets of a bilayer expand and contract independently from one another. We will
have occasion to call upon this concept later on.
E.rpc>ritnmt 3:[291A vesicle composed of pure D M P C was
shown to go from a spherical shape to an invaginated state and
then to a two-vesicle form (Fig. 9) when the coverglass, resting
Fig. 9. Effect on a DMPC giant vesicle when the coverglass is lightly tapped 1291.
A n g m . C h ~ mInr.
. Ed. E q l . 1995, 34, 209 1-2106
Cytomimetic Organic Chemistry
REVIEWS
on the sample. was lightly tapped. It was correctly argued that
this experiment demonstrates the metastability of vesicle morphologies. One important question was not addressed however:
Is the shape rransformation (in this case triggered by mechanical
agitation) secn frequently or is it a rare occurrence? We pose this
question because in our experience many bizarre morphological
events occur only infrequently, so that publishing their pictures
without some statement as to their “probability” can be misleading.
The study of the physically induced vesicle transition is instructive for another reason. Vesicle preparations in experiment
3 were examined as thin films placed between a slide and a
coverglass. We have avoided handling samples in this manner
for two reasons: a ) It is desirable to minimize vesicleiglass interactions espccially those that might crush vesicles between two
glass surfaces (recall that GUVs can have diameters of 0.2 mm
or larger; scc Table 1 ) . b) We planned to manipulate our vesicles
( i t . move them about, inject into them etc.) much the way
cytologists handle living cells. This precludes the use of a coverglass. Since uncovered thin films evaporate quickly, we invariably used a thick aqueous sample placed within the confines of
a circular “wall” (details to be given later). A more sophisticated
setup that accomplishes the same thing is given elsewhere.[311
E.::\/icuirncwr1:[3’1
Shape transformations of DMCP GUVs in
ion-free water were studied as a function of temperature
(Fig. 10). The morphologies portrayed in Figure 10 occurred in
F-
0
Fig. 1 1 . Apparent thape changes in phase-contrast microscopy t h a t :wise from
rotations rather than from true rnorphoio_eical changes
The ingenious studies of giant vesicles by Evans and Needcan serve as a fitting conclusion to this historical
section (which is by no means intended to be an all-inclusive
review). Evans and Needham aspirated a 20 pm GUV with a
5 pm micropipet under a suction pressure of 0.03 atm or less.
This caused the vesicle to be partially
drawn into the pipet (Fig. 12) with a
concomitant increase in the membrane area (as measured indirectly
from the membrane projection
length inside the pipet), Two types of
I’ A\piratlon Of
a
2Opni C;UV i n t o a 5 pm
27.2”C
tie
36.0T
39.1”C
40.9T
41.0”C
10. Shape tra~ishrmationsofa DMPC GUV in ion-free water n s a function of
tempcfiitiire [3l I
35 out of 98 vesicles when “lateral stress” (of uncertain definition) was exerted upon the vesicles prior to heating. The GUVs
changed from spherical (27.2 ’C) to an ellipsoid (36.0 “C) to
various pear-shapes (37.5 -40.9 ~ ‘ C )All
. these morphologies
were stable and reversible. A further temperature increase of
0.1 ’ C to 41 .O ‘C destabilized the pear-shape and created what
appears to be an attached “bud” on the verge of becoming a
separate daughter vesicle. Even more exotic transformations
were observed but will not be detailed here. Suffice it to say that
the shape transitions are qualitatively explainable in terms of the
Svetina--Zeks modelr3”]in which the two component leaflets of
the bilayers have slightly different thermal expansivities.
A degree of caution is necessary whenever vesicle morphologies are studied. It must never be forgotten that phase-contrast
microscopy detects only a thin band around the equator of the
vesicle. Consequently. it is possible to confuse a “sphere-topear” transition with a 90,’ rotation of a pear-shaped vesicle
(Fig. 11 ) . Similarly. a 90’ rotation could be mistaken for a transition from a n invaginated vesicle to a “vesicle inside a vesicle”
(Fig. 11 ). Such ambiguity can be resolved by demonstrating the
reversibility o f the transition; it is unlikely that 90.’ rotations
coordinate themselves with medium conditions as the latter
switch back and forth between two states.
experiments were performed: a) The
pipet hlnncr a suction pressure 01 0.03 alm o r less (321.
suction pressure was increased at
constant temperature. b) The temperature was increased at a fixed suction pressure. These experiments provided two thermoelastic coefficients: the cotnpressibility modulus K and the thermal expansivity x . A few specific
results. cited below, illustrate the power of the method.
When a GUV is cooled at constant pressure from above to
below the phase-transition temperature, the membrane contracts abruptly by about 20 %. In addition, the compressibility
modulus increases dramatically. For example. the K value for
DMPC (with a transition temperature of 24 ‘ - C )increases from
1 5 0 d y n c m - ’ a t 2 9 ” C to855 dyncm-’at 8 ’ C . Addingequimolar amounts of cholesterol to the DMPC at 29 <‘Cincreases the
K value from 150 dyncm-’ to 685 d y n c m - ’ even though the
membrane remains fluidlike (i.e. in its liquid-crystalline phase).
Cholesterol apparently forms a tight complex with DMPC that
greatly reduces the bilayer compressibility relative to a bilayer of
pure DMPC above its transition temperature. Evans and Needham were also able to detect the presence of a “rippled membrane phase” from an unusually short projection length in the
mircopipet at low tensions. A ripple phase has a corrugated
structure with the lipid chains lying perpendicular to the overall
membrane plane.
As is evident from the references cited thus far, the great
majority of the giant vesicle work has appeared in the biochemical and biophysical literature. Chemists, especially organic
chemists such as ourselves, have paid comparatively little attention to giant vesicles. In attempting now to publicize the topic to
the chemical community, we face a severe communications
problem, a problem stemming in part from the fact that giant
2097
F. M. Menger and K . D. Gabrielson
vesicle research is a composite of chemistry and biology. Whereas chemistry contributes its simple and defineable experimental
conditions (composition, concentration, etc.). biology contributes a particular brand of data reporting (photography). As
a result of the latter, chemists are (at least for the moment)
denied the graphs, tables, equations, and spectra they love so
well. Nonetheless, we ask that chemists resist exclaiming “This
is not chemistry!” What else but chemistry could it be when, for
example, a giant vesicle, prepared from a compound purchased
from a chemical supplier, divides in two after being exposed to
a synthetic surfactant? Eventually, of course, we will yield to
reductionism and discuss our “chemical systems”[’] on more
molecular terms. But before this can be accomplished, the fundamental behavioral modes of our membrane models must first
be observed and recorded. Occasionally, explanations will be set
forth here at the molecular level, but the reader will likely find
them rather primitive. Remember, however, that we are at an
early stage of understanding and that, at the moment, many
problems in vesicle chemistry are. as J. Wiesner would say, “just
too complicated for rational, logical solutions. They admit of
insights, not solutions”.
4. The Light Microscope
“The ladder of natural systems consists of elementary particles. atoms, molecules, macromolecules, cell parts. cells, organs. organisms, populations, and ecosystems” (R. Wesson). In
the main, elemental particles have been the province of physicists; molecules the province of chemists; macromolecules the
province of biochemists; and cells the province of cytologists.
This division of labor is becoming increasingly obscure. Inevitably, as a particular level of science matures, the adventurous seek out the next higher level of complexity. Those who d o
so are of the same temperament as Thomas Lincoln who once
remarked to his son (Abe) that people should move their homes
as soon as they can see the smoke from their neighbor’s chimney. Chemistry provides as good an example as any of this
relentless move up the complexity scale. Thus, one hears more
and more at chemistry conferences about DNA, antibodies. and
receptor sites. The present paper recommends that chemists take
a two-step jump from molecules to cell parts. A recommendation is hardly necessary because chemists have already applied
their familiar methods (NMR, ESR, kinetics etc.) to membrane
systems. What is unusual. however, is the suggestion that
chemists befriend that old workhorse of the cytologist. the light
microscope.
Those who have a mere passing interest in the subject of
cytomimetic chemistry will wish to skip this section on light
microscopy and move directly to the subsequent results section.
But those who might be tempted to actually enter the field
should read the following brief remarks on the light microscope.
The light microscope has, in recent times, been upstaged by
other techniques, but “it is not widely known outside the biological community that a renaissance and a revolution in light
microscopy are now underway”.[331
Our laboratory is equipped with two instruments: A Leitz
Laborlux S standard light microscope and a Nikon DiaphotT M D inverted light microscope. The latter style, possessing the
2098
lens assembly below the stage and illumination above it, allows
for easier micromanipulation of the vesicles (discussed later)
and for less interference problems when water condenses on the
coverslips at reduced temperatures.
Both microscopes are fitted with 10 x ocular lenses. The objective lenses have four recommended features: a) They are
specifically manufactured and sold for phase-contrast work. b)
They are flat-field or P L A N 0 to reduce the curvature of the
field and thus bring into focus both the central portion and
periphery of the image. c) They are achromatic which means
they are corrected for one wavelength (yellow-green) with respect to spherical aberrations. Owing to this feature, a green
filter is always placed in the path of illuminating light source. d)
They are long working-distance lenses as required for our thick
vesicle samples. The four features, specified when purchasing
the lenses, are not recommended for high-quality bright-field,
cross-polarizing. or fluorescence microscopy.
The microscope condensers, which focus the light onto the
sample. are universal in the sense that they can handle many
forms of light microscopy. Since their iris is always wide open in
phase-contrast microscopy, the condensers are never touched
once they are initially centered and focused.
Each objective lens is associated with a numerical aperture
(NA) at the time of its manufacture. Our NAs vary from 0.25 to
0.55. Two important parameters, the resolution and depth-offield, depend upon the N A value. Resolution can be defined as
the smallest distance between two points that can be distinguished. According to the Rayleigh equation,r341the greater the
NA. the better the resolution. Note that objects smaller than the
resolution can be detected but not in any detail. Resolutions of
our various objective lenses vary from 0.52 pm to 0.87 pm.
Depth-of-field can be regarded as the distance from the
nearest image plane to the farthest image plane that remains in
acceptable focus. Depth-of-field can play a role, for example, in
the study of a small vesicle departing from the inside of a larger
one. As the depth-of-field diminishes. it becomes increasingly
unlikely that the small and large vesicle will reside in the same
image plane and thus both be in focus. Although a large N A
enhances resolution, it decreases the depth-of-field, thereby
making focusing more troublesome. For example, N A = 0.25
and DF = 8.5 pm; N A = 0.55 and D F = 1.5 pm. Working with
light microscopes teaches the principle of compromise.
The light microscope can be used with an array of optional
peripheral equipment (Fig. 13). Special mention should be
made of a solid-state camera that is inserted into a microscope
port made for that purpose. The camera’s signal is sent to an
Argus I0 image processor that digitally enhances images to give
greater contrast. It also provides a “zooming” capability so that
a 400 x magnification becomes an 800 x or 1600 x magnification. The block diagram in Figure 13 also includes a VCR for
making video tapes in real time, a monitor for viewing images
on a screen, and a printer for making “stills”. One should not be
intimidated by the apparent complexity of Figure 13. All components are commercially available, attached quickly to the system, and easy to use.[351Simple refocusing and stage repositioning are the main skills necessary to track “unidentified
swimming
One of the major advantages of GUVs over their submicroscopic cousins is that they can be micromanipulated just like
A n p w . Chem. Int Ed. EnRI. 1995, 34. 2091 -2106
Cytomimctic Organic Chemistry
Image
REVIEWS
BIW Monitor
Panasonic
Microimage
oration and surface vibrations of the sample. Of course, when
micromanipulation was desired, a coverglass was not possible.
A simple homemade thermostated stage was installed when temperature control was necessary.
This concludes the background information pertinent to the
subject of cytomimetic chemistry. Individual tastes and needs
will dictate whether too much or too little detail has been provided. Either way. the time has arrived to inspect actual photomicrographs and view first-hand the intricacies of membrane
behavior. Most of the photomicrographs in the next section
have not been published previously.
5. Experimental Results
Fluorescent
Microscopy on
Leitz
Leitz
1
Nikon Picainjector
Fig i 3 Block di‘igraiii 01’ video microscope setup and peripheral equipment used
Many GUV experiments can. however. be carried out with a simple
micro\c~ipecqiiipped with a phase-conIrast attachinenr and a Polaroid camera
i n O U I -w o r k
cells. Micropipets as small as 1000 8, in diameter can be made
with it commercially available “pipet puller”. A vertical glass
capillary. weighted at the bottom end with suspended metal
blocks. is threaded through an electric coil at the top end. When
the switch is turned on, the hot coil melts the capillary section
within i t , the weights come crashing down, and the capillary is
pulled inlo ii narrow-diameter pipet. The pipet can be further
fashioned with a microgrinding wheel or with a so-called microforge. The latter allows one to polish the end of the pipet o r to
bend i t into any desired shape.
Micropipets were useful to us primarily for holding vesicles in
placc and for injecting into them. Holding pipets have large
smooth ends that can latch onto a GUV when a slight suction
is applied. Injection pipets are much smaller in diameter than
the vesicles and are ground to a sharp point. Administration of
both suction through an empty pipet and pressure on a filled
pipet was carried out with a “picoinjector” hooked to a tank of
nitrogcn. A picoinjector can supply orders of magnitude less
liquid than the nanoliter volume of a typical GUV.
Micropipets were directed toward a desired point in the microscope‘s image with the aid of a micromanipulator. This
device. fixed to the microscope’s stage, has a “joy-stick” that
provides cxtreinely small movements to the end of an attached
pipet. All the micromanipulation techniques described here are
commonplace in cytology.
Mention has already been made of sample preparation by the
DDAB hydration method (see Section 2.4). The hydration took
place within ;I rubber washer (14 mni id.) cemented onto a glass
slide. Sufficient sample (0.45 mL) was used to fill the washer and
to minimize the amount of air between the sample and the
coverglass resting upon the washer. Coverglasses reduced evap-
Our experimental results have been categorized below into
various cytomimetic processes (aggregation, fusion, fission,
etc.). Each process is portrayed by a series of still photographs
selected from video films produced by the microscope setup.
Admitedly, viewing the still photographs is like looking at a
Mickey Mouse film one frame at a time. Fortunately, however,
vesicle behavior can in fact be reasonably well described with
still photographs as long as they span a suitable time period.
This is not to say that nothing has been sacrificed by the unavoidable shift from a dynamic to a semistatic representation.
No still photograph can, for example, elicit the delight of seeing,
in real time, an entire vesicle fusion or fission. And no still
photograph is likely to instill a sense of wonder of the type that
once caused a visiting scientist to exclaim: “How do you make
those cells d o that?” Those “cells”, we then revealed to the
visitor, had been made from a compound purchased from Eastman Chemicals; and they d o what they do bccause of simple
chemical and physical stimuli.
5.1. Hydration
We have already described how hydration of lipid films leads
to the production of giant vesicles (see Fig. 2). In our hands, this
method led mainly to multilamellar vesicles (which is one reason
why the solid-state hydration, pictured in Figure 3. was developed). It turns out that giant multilamellar vesicles, prepared by
the drying-rehydration method, also tend to form unilamellar
vesicles if allowed to sit around and hydrate for several days. This
hydration process was examined under the microscope (Fig. 14).
Fig. 14. Phase-contrast photomicrographs of ii giant iiiultilainellar vesicle hydrating to give, ebentually, small unilaincllar vesicles. Left: after 2 5 h Right: after 4.5 h
Scale-bar in the bottom-right of photographs = 100 pm.
2099
F. M. Menger and K. D. Gabrielson
REVIEWS
Multilamellar vesicles are seen first to “blister” at their outer
shells. These blisters then grow into tubules that project radially
from the vesicles’ outer surface. Ultimately, the tubules break
off to become unilamellar vesicles. Thus, one-by-one the layers
of the multilamellar vesicles are eroded into GUVs. The apparent instability of multilamellar vesicles relative to their unilamellar counterparts may stem, in part, from electrostatic repulsion
between the concentric DDAB layers within the onionlike configuration.
5.2. Aggregation
A. Einstein once wrote, “The only apparatus necessary for
observing Brownian motion is the microscope, and it need not
even be a particularly good one“ Similarly, a simple microscope
is all that is required to visualize vesicle aggregation. One
merely adds low levels of
Na,SO, (l.OmM) to a sample
containing widely spaced giant vesicles. Over the course
of several minutes, the vesicles
will assemble into clumps
(Fig. 15). Note that this experiment, carried out early on
in our research, used darkFig. 15. Dark-field photomicrographs
field microscopy rather than
of sulfate-induced aggregation of githe phase-contrast microscopy
ant vesicles. Prior to addition of sulthat we now prefer.
fate, there were only three vesicles in
the microscopic field.
The aggregation process is
viewed as an electrostatic neutralization of the long-range double layer repulsion among the
~ e s i c l e s . ~ Dianionic
”~
sulfate can, apparently, serve as a far
more effective “adhesive” than can the bromide counterion of
DDAB (Scheme 1).
Scheme 1
N o sulfate-induced fusion was seen with the GUVs although
this has been reported with vesicles only 80 nm in diameter.[381
Small vesicles have a high curvature and might be expected
to show a greater propensity to fuse than the GUVs. One is
reminded of the so-called “Ostwald ripening” in which large
colloidal droplets grow at the expense of small ones.[391Interfacial tension within the small droplets drives the coalescence.
The absence of fusion with the sulfate-treated GUVs suggests
that sulfate is unable to bring the vesicles sufficiently close
together to overcome the “hydration repulsion” as would be
necessary for fusion. Hydration repulsion reflects the need to
desolvate headgroups prior to phase instability in the membrane.[“’]
2100
5.3. Fusion
I f it were not for barriers to membrane fusion, humans would.
like slime mold, exist as multinucleated masses of protoplasm.
And if it were not for mechanisms that trigger fusion, many
important biological events (infection, fertilization, conjugation
etc.) would not be possible. Little wonder that fusion is a recurrent theme in treatises on membrane behavior. Unfortunately, a
footnote number is the only homage we can give to the immensely fertile literature on membrane
Space restrictions force this embarassingly rapid trek through the subject of
fusion in which the focus lies entirely on our own cursory experiments.
5.3.1. Physical Constraint
Two giant vesicles can be forced into intimate contact with the
aid of holding pipets under slight suction (Fig. 16). Note that in
Fig.16 A failed attempt at physically inducing fusion in two DDAB giant vesicles.
The vesicles were maiiipulatrd by inriins of holding pipetr uiider slight suction. In
thc left photograph. the vesicles have just been pulled apart. an action that caused
slight distortion iil the lefthand vesicle.
the left photograph, the lefthand vesicle becomes distorted by
the pressure from its neighbor. Moreover, when the vesicles are
separated, the lefthand vesicle in the right
photograph is pulled, however slightly, into an oval shape. The shape then restores
itself instantly into a sphere. Despite the
obvious close proximity of the vesicles, no
fusion occurs for periods up to an hour.
Fusion can be induced chemically (as we
shall see next) but not physically.
5.3.2. ,4cetate-lnduced Fusion
A remarkable sequence of events was observed when 100 pL
of 0.25 M NaOAc was added, in tiny portions without stirring. to
a sample of giant multilamellar vesicles (0.45 mL of 9 x 1 0 - 4 ~
DDAB; drying-rehydration method). First. the vesicles began
immediately to disintegrate. In the course of 20 minutes, the
vesicles deteriorated into structureless masses that eroded into
submicroscopic material to give, finally, an absolutely clear microscopic field. (This vesicle disappearance was anticipated because didodecyldimethylamnionium acetate is a highly watersoluble compound that does not form giant vesicles). But a
peculiar thing happened when the samples were kept at or below
REVIEWS
Cytomimetic 01ganic Chemistry
the DDAB phase-transition temperature of 17 "C. Small *'globules" reappcared and began to speckle the microscopic field.
The term globule is used because the structures were too opaque
to be iiiultilamellar vesicles. Whatever their exact morphology,
the DDAB globules fused at a prodigious rate. Figure 17 shows
4
-
t
\
,
/
*
--I/'
'
~
\+
i'
._
,
,
/ /
,
'
Scheme 2
5.3.3. DPA-Induced Fusion
Engberts et al.[44]have reported that the dianion of dipicolin-
Fig 17. l'li~is~-~~)iili-~i,t
micrographs o f a n acetate-induced fusion taking place over
k u wcoiid\ Scalt-bar = 2.5 Lcm.
ii
a sequence in which two globules, after having resided side-
by-side for many minutes. suddenly fused over the course of
u fen. seconds. The fusion was reproducible in several experi-
ments.
Onc cannot attribute the vesicle disappearance solely to 0smotic strcss since the effect was salt-dependent (being caused by
iicctate. fluoride. and hydroxide but not chloride). Two notions,
however satisfactorily explain our observations : a) Strongly hydrated anions, such as acetate, bind relatively loosely to cationic
surl',lces.['?l b) According to the Svetina-Zeks model,[301the
couplcd leatlets in a bilayer can act independently. Thus. when
excess x e t a t c is added externally to the DDAB vesicles, acetate
exchanges with bromide to produce an outer leaflet that is more
highly dissociated from its coirnterions. Owing to the resulting
hendgroup headgroup repulsion. the outer leaflet expands relative to thc inner one. Such asymmetry would be expected to
incrcnsc c ~ i t - v i i t u r eand.
~ ~ ~therefore,
]
promote the expulsion of
small vcsicics and the eventual disappearance of microscopically
visible structures (Scheme 3).
(
/ii,ni
liif
E d 01q/. 1995. 14. 2091 -2106
Fig 18. Phase-contrast micrographs of a DPA-induced fusion of DDAB vesicles of
unknown strncturc. Scalc-bar = 50.0 FirI. Process took p l ~ c cin less than four
scconds.
2101
F. M. Menger and K. D. Gabrielson
REVlRlVS
Fig 19. Effect o f DPA on DDAB GUVs Top left 10 top right 10 min. Top right
to bottom lelt: 1 11. Bottom lelt to bottom right: 1 h.
that the DPA incorporates itself into the headgroup region
and spreads the lipid molecules apart (Scheme 3). Since
the lipid packing is thereby
loosened, distortions from the
usual spherical shape become
Scheme 3
feasible. A second possibility
relates to the fact, often observed in our work. that the GUVs
seem to be under osmotic tension (i.e. internal pressure that
inflates the vesicles). The source of the tension in the absence of
any added salt is not yet understood. In any event, the DPA may
render the vesicles permeable to water and small molecules. thus
allowing pressures to equilibriate and vesicles to deflate.
5.4. Endocytosis
In many experiments with DPA, we saw an occasional GUV
undergoing a process remarkably similar to cellular endocytosis
(Fig. 20). A giant vesicle begins to engulf a smaller vesicle, and
after about 30 minutes the smaller vesicle is completely “eaten”.
No doubt the DPA promotes the endocytosis by enhancing the
membrane flexibility. One wonders what sort of endocytosisstimulating substances are produced by an amoeba when it senses an edible morsel in its vicinity. Or how a virus manages to
persuade a host cell to allow it to enter.
By now it should be obvious that a pattern is developing. We
are enumerating wonderful morphological changes in a simple
nonbiological membrane. But the readers are not being supplied
with the molecular basis of these changes except at the most
rudimentary level. There should never be an apology for the lack
of detail when an area of science opens up and the investigation
has only touched the surface. The pleasure of discovery in science derives not only from an incisive analysis but from the
formulation of a problem which, according to A. Einstein, is
2102
I;ig. 20. Giant unilamellar vesicle undergoing a DPA-induced endocytosis over the
course of about 30 min. Process was secn repeatedly but with only the occasional
vesicle.
“often more important than the solution”. New observations
leading to unanswered questions, unsolved puzzles, fresh possibilities, and untried experiments-that is what this paper is all
about. In essence, we have taken the advice of R. Hoffmann:
“Row a boat into that tall mysterious grass just because you
have heard a strange bird call there”. In our case, the “mysterious grass into which we rowed” is the world of chemical microscopy, and the “strange bird calling us” is the giant vesicle
with all its amazing transformations.
5.5. Fission
Fission was of particular interest to us because the combination of fission, growth, and fission again is equivalent to selfreplication, a long-range goal of ours (Scheme 4). As will be
shown, fission has indeed been achieved but not the “one-vesicle-into-two-equal-vesicles” as would be ideal for a self-replicating process.
fission
0+o
I
I
Scheme 4
growth
C y t o mi in c t i c 0rga nic C heinis t ry
REVIEWS
5.5.1. Mechanical Agitation
Figtirc 21 represents an attempt to inject a sharp micropipet
into ;I vesiclc being held by a holding pipet under slight suction.
Thix wab ; i n early cxperiment. and at the time wc did not realize
that the giant vesicle was multilamellar. In any event, the injecting pipct (being controilcd by a micromanipulator fixed to the
Fig 21 Thigm.itrnpic f i s i o n 111 nhich prodding ol' :I multiliiinellar vesiclc c;itIsei
tlic I ' ~ i ~ i n . ~ :tiii oi d~ICIC:IIC
i
o l i i smaller vcsicle. The parent \csicIc is held 111 place h)
,I hOldlll:L
;I , li~ ll. ppiPC'- conlr~,llsl,
bq :I i~lc~omaniplil,lt~r, prod, thLI
I.C\I<~lC \url:Lcc
,,
the
microscopc
experienced great difficulty in
vesicle. Eventually. the prodding caused a new smaller vesicle to
forin. Wc surmise that the outermost bilayer of the multilamellar vesicle had been physically damaged by the pipet. Lipid
molecules i n thc injured bilayer then had an opportunity to
reorganize themselves into an independent unilamellar vesicle
where bilaycr-- bilayer repulsion was not a factor.
Occasionally. one encounters an oligolamellar vesicle in
which the concentric bilayers are sufficiently well-spaced that
thcy ;ire visible individually. Figure 22 shows the layer-by-layer
destruction of :in oligolamellar vesicle when agitated by a charp
pipet. The vesicle becomes sinaller and smaller a s the layers are
shed.
5.5.2. Dilution
When giant multilamellar vesicles are prepai-cd by hydration
o f a lipid film (a method rhat. as mentioned. was supplanted by
our solid-state hydration) the vesicles remain unchanged for at
least a day unless the sample is diluted four- to twentyfold with
water. Dilution stimulates the ejection of 5 S 3 0 pm GUVs from
the multilainellar vesicles until the latter ha\ e entirely disappeared (Fig. 23). As small GUVs depart, their inner contents are
Fig. 23 Ejeclion o f a small unilaiiicll;ir vesicle froin ii ~niiltil:i~iirIlar
vcsiclc induced
by dilution. Note how the particul;ite niatlcr insidc the iiniliiincllar vesicle 15 expcllcd
throurh ii defect created zit thc contact point d t h e !\w w\icle\. T h e t \ w photozr;iphs are apaced three seconds iipdrt. Less tlian live scconci< Iatur. the daughter
\TsicIes had mo\cd :iwiiy from thc parent a n d healed thc del'cct.
rapidly expelled from the hole where the GUV and multilamellar vesicle had been momentarily attached. (The hole then heals
immediately to give an intact GUV). Expulsion of the internal
contents of the GUV is a further indication of the internal stress
that seems to characterize GUV systems.[""]
Why does dilution with water induce fission'? Osmotic stress
does not seem to supply an answer because the vesicles. prepared in ion-free water, were also diluted with ion-free water. It
seems. instead, that there is a delicate balance between vesiclcvesicle repulsion and bilayer- bilayer repulsion. When a sample
is diluted, the vesicle-vesicle repulsion is diminished, but the
bilayer- bilayer repulsion within the multilainzllar vesicles remains unaffected. Consequently. GUV formation becomes
thermodynamically favored. Small energy diffcrenccs between
different morphologies are key to giant vesiclc behavior.
5.6. Birthing'"]
} ig 1 2 I l ~ c \ ~ y l c \ l I:i)crs
~il\
ofL1nollg0I;1l~1ell~lr
gl;lnr V C ~ & hclllg ruptlll-ed \)y
pipet. C ~ I I N I Itlic
~
~.s\icIe10 becomc viialler and milllei-.
sl1ai.p
The interactions between surfactants and GUVs attracted our
attention owing to the widespread use of surFactants in solubilizing biomembrane component^.^"'^ M. N. Jones'18' proposed
that surfactants bind to phosopholipid membranes by means of
hydrophobic forces. When saturation levels are reached, the
bilayer begins to expel mixed inicelles composed of lipid plus
surfactant. Ueno and Akcchi.[4'1 who observed 200-nm vesicles
by freeze fracture electron microscopy. found that octyl glutransfornls a vesicle into "
Of
30-n1n spheres
(resembling a raspberry). These tiny particles dcpart one-by-one
F. M. Menger and K . D. Gabrielson
REVIEWS
from the surface to become, soon thereafter. solubilized in the
water as mixed micelles. We know of no example in which vesicle
destruction by a surfactant has been obscr-ved by light microscopy.
Adding 25 pL of 5mM octyl glucoside to a standard GUV
preparation (20-21 ' Cj, giving a surfactant concentration well
below its critical micelle concentration of 2 5 m ~ caused
,
minimal vesicle destruction in the first hour. The most striking observation was a "birthing process" involving the occasional GUV
that possessed a smaller vesicle within it. Under the influence of
octyl glucoside, the entrapped vesicle can force its way through
the bilayer wall of the parent vesicle (Fig. 24). A gap is created
in the parent vesicle that heals instantly (bottom photographs).
The sequence of events was observed repeatedly with every
preparation. Light microscopy can obviously capture dynamic
events that are difficult to detect by the more commonly used
electron microscope. Moreover, artifacts from drying, metal
coating, etc.. a problem in electron microscopy. are avoided.
Combining the results from both light microscopy and electron
we surmise that octyl glucoside weakens
the cohesion among the organized DDAB molecules, perhaps
by the formation of weakly associated spherical particles. The
membrane subunits are able to temporarily separate from one
another and, thereby, allow the passage of an entrapped vesicle.
Evidence for this model came from experiments using higher
octyl glucoside concentrations (10-25 mM) that rapidly destroy
the GUVs. During the process. 0.1 - 1 .O-pm particles are seen to
speckle the vesicle surface (not shown). These particles (which
appear from bright-field microscopy to be either solid o r multilainellar lipid) break away while, simultaneously, the diameter
of the G U V diminishes a t a rate dependent on the octyl glucoside concentration.
5.7. Foraging14]
Cy t om I inc t ic 0 rgan ic Chemistry
The foraging vesicle grows in diameter with each small vesicle
that is consumed. When small vesicles no longer exist in proximity of‘ the foraging vesicle, an unexpected and previously unreported cvent occurs: The large vesicle disintegrates by a sequential cjcction o f lipid molecules (Fig. 26). a process that takes
REVIEWS
silience to internal pressure. Or a perturbing solute might be
introduced to determine how its effects on the interior and exterior leaflets of the bilayer compare.
Injection was not an easy thing to achieve. I n the case of
multilamellar vesicles, attempts to inject led to .;bedding of the
bilayers (see Figs. 21 and 22). Most unilamellar vesicles. on the
other hand, burst when prodded with a micropipet. We finally
succeeded. however, with GUVs that are 2 200 p i in diameter
containing 5 % cholesterol. Such vesicles are pliable, and they
resist bursting. A good example is shown in Figure 27. It is
Fig. 27. Insertion o f B micropipet into a giant unilamellar \csicIc Thc \esiclc
composed of DDAB and cholesterol (95:5). Scalc-bar = 50 pin
IS
believed that the pipet has actually penetrated the bilayer because the vesicle becomes distorted in the experiment and because both the tip of the pipet and the vesicle are in focus. A
further indication of pipet entry into the vesicle interior is given
in Figure 28. A bright particle within the vesicle i s removed by
the suction applied through the pipet. Note also the easy deformability of the vesicle as well a s its ability to heal the wound
created by the pipet.
5.9. Final Remarks
Fig. X . I)ec.i! <,I p n t uiiiliiniellar vesiclc immediately following tlic forasing yeen
in 1 IF. 2 5 . 1 i i t i i c diw>lulion of thc memhranc takes cix seconds
only ii few scconds. It is as if a defect in the GUV created an edge
of exposed lipid molecules. Lipid molecules are then continuously solubilized from this defect by the cholic acid until the
vesiclc hilaycr disappears. Remarkably, the original spherical
shape i s retaincd throughout the process. The products of the
membrane dissolution are submicroscopic and may well be the
mixed micclles postulated by others.[4R,4 y 1 At higher concentrations. crystals of DDABkholate eventually fall out of solution.
It should bc noted that another steroid, cholesterol, has no such
effect on thc G U V s . In fact. 30% cholesterol incorporated into
the DDAB bilayer actually stabilizes the vesicles.
5.8. Injection
Injcction into giant vesicles has been a high-priority objective
of ours sincc the very beginning. If a pipet could be inserted into
ii vesicle (thc way cytologists routinely do with living cells). a
host or expel-iments fall into place. For example. biological
macroiiiolccules. such as enzymes and DNA. could be examined
within the confincs of a giant vesicle. Or various volumes of
water might he injected into a vesicle to test the bilayer’s re-
We finish, appropriately enough. with a quote from Albert
Szent-Gyorgi in h i s “Personal Reminiscences”:
“In my hunt for the secret of life, I started research
in histology. Unsatisfied by the information that
cellular morphology could give me about life.
I turned to physiology. Finding physiology too
complex, I took up pharmacology. Still finding
the situation too complicated. I turned to bacteriology.
Biit bacteria were even too complex, so I descended to
the molecular level, studying chemistry and physical
chemistry . After twenty years’ work. I was led to
conclude that to understand life we have to cicscend
to the electronic level, and to the world of wive
mechanics. But electrons are just electrons. mid
have no life at all. Evidently on the way I lost
life: it had run out between my fingers.”
The above words should not be regarded as a call for biologically oriented chemists to abandon their craft. Quite the contrary. their efforts should be intensified. But this should be done
while bearing in mind that the living cell has an organizational
complexity totally absent in the reaction flask. Future emphasis
must. therefore. be away From ”single-moleculc chemistry” and
2105
E M . Menger and K . D. Gabrielson
REVIEWS
171
[XI
(91
1101
[II]
[I21
K.Bctltner. P l ~ ij i ~ i C/i<'!iii.\lrj
l
Of Lir;ii,y Ti,\.\i/~\~ i i Life
d
Pwi'
a n d Wilkcns. Baltiinorc, 1933.
A . E. Smith. D. H . Kenyon. Pcw\/w(riii,\iii B i d ~ii~i/:Ifi~i/.
1972. 52')
A D B;inghaiii, M . W. Hill. W G. A Millcr, , L f r i / i . Monh Eiiil. 1973. 1. 1.
Li/io.wiiirs (I,\ Drii,y C ' w r i m ( E d . : G Gregoriadis). Wile>. Winchellei-. 1988
Lf/moiiii,\, (td : M. J O s t r o ) . Dckker. New Ywk. 1987 G . Storm. H P
W i l m s . D .I A . Crominelin. BiiU/i?ro[jj, 1991. 3. 2 5 . G L ScherphoT. T. Daemen, I3 S. H Spaiijcr. F. tl. Roel-dink. L@d\ 1987. 22. X9l. S Wright. L.
Huang. ,,!(/I f l r i r , ~ Dc/.
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Rc.1.. 1989. 3. 343.
R. R. C. New. L i l ~ o \ o i i i c \ .:I frui,rii,d A p p r o d i . fKL Press. Oslbrd, 1990.
F. M . Mengei-. I-J. Lee. P. Aikena, S. Davis J, Collotd /iiriv/ii( i' Sc i . 1989. 12Y.
1x5
[I31 J. M. H . Kreiiiei-. M . W, J. Esker. C. Parhmaiiiniioliaran. P.H . Wierscnia. Bioi , / i i v i i i , \ l r t ,1977. /(i.
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1141 D . W. Deamcr. A D. Bangham, B i i i i ~ / i ; / i Biop/ij,\.
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A r r i r 1976. 443, 629. M. J
Blandamer, B Briggs, P. M. Cullis. J B. F. N. Engherts. D. Hockstra. J C / i i w i .
SW Fiiradw 7 k ! i i . \ . 1994. 90. 1005.
[I51 F M. Mengcr. D. S. Davis, R . A. Pcrsichctti. J-J. Lce. J. , . h i . C/ieiii. Soc. IYYO.
112. 2451.
[I61 J. P. Reeves. K. M. Dowben. .I Ccil. P/ii~.\;o/ 1969. 73, 49;H . H. Huh. U
Zimrnernian. H . Ringsdorf, F E E S /,IJII. 1982, 140, 254.
[ i 7 ] D. Ncedhmi. E. ELans. Eioi~/iwii.\rri1988. 27, 8261.
[ I X ] N . Oker. J. f; Scheerer. R . C. MacDonald. Bfoi,/iiiii b ' i i ~ p / i i . \ .A < . ~ r 1982.
i
6Y7.
3x4.
[19] N Oker, R . c'. MacDonaId. B i ( ~ ~ / i i iBiop/ii.\.
ii.
Acto 1983, 734. 54.
[ X I K.Higashi. S. Suruki. H . Fujii. Y. Kirino. .I Eiochei~i.1987, 1111, 433.
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[24] H . T. Tien, B i I q w Lipid ;Mmit/?riii~rs( E L M ) . T/iiwrj i i i i i l Prui~liw.Dekker.
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YS.
Fig. 28. Withdrawal o f a particle (bright spot) from tlie interior ofil ginn1 utiiliiiiicI1,'ii - \csicIe
,
(DDAB + cholesterol 95 5 ) using ;in in\ei-tcd mici-opipet under \light
s uct io 11.
towards molecular assemblies. Because an organized group of
molecules behaves differently from a inolecule operating as a
single entity, there is a need to define the differences. It is with
this philosophy that our research group has worked over the
past two decades;"] and it is with this philosophy that "cytomimetic organic chemistry" was conceived.
It was a novelist, Tim O'Brien, who wrote the following
words: "The thing about a story is that you dream it a s you tell
it, hoping that others might dream along with you, and in this
way memory and imagination and language combine to make
spirits in the head". Perhaps this is also the best that scientists
can hope for: to create spirits in the mind.rsO~sll
wx.
[25] P. Mueller. T. F. Chien. B. Rudy. Biop/ir\. J . 1983. 44, 375. For another example o f dark-field microscopy. see H . Hot;iiii, .I , V / d Eiol 1984. 178, 1 I 3
[26] R. Buschl. H . Ringsdorl: W. Zimmeriiiiinn, FEES Lrrr. 1982, 150. 38.
[27] H . Ringsdorf. B Schlarh, J. Ven7iiicr. A i i , y r w . Chciii. 1988. IfiO. 117 Aiigiw
CIiiwt. t i i / . E d . EngI 1988. 27. 113
[?XI E. Sackmann, H-P. Duwc. K . Zenian. .A. Zilker in S i r i i c r i i r c ~ i i Dj.iiriniic.\
d
O/
A ' I K / I % A d , Piorerii.s, i i i i d ;Miwhriiiics. (Eds.: E. Clenicnti. S. Chin). Plenum,
Ne\v York. 1986. p. 251
[29] E Sackinann. H-P. Duwe. H. Eiigelhardt. Fm~iduif l i s m . \ \ . Climi. Soc. 1986,
81. 2x1.
[XI] S Svetina, B Zeks, B i o m d Rfoi~hoii.,l<,ru 1983. 42. 86.
1311 J. Kiis. E. Sackiminn. Biriplij~s.J . 1991. 60. 825.
[32] E. Evans. D Necdham. F i i r d i i j D r \ i ~ i i . s s . C / i w i . S i x 1986. XI. 267.
[33] D. L Taylol-. M . Ncderlof. F. Lanni. A. S.Waggoner. A i i i w Sciivi/rvr 1992. KO.
322
(341 I\. J L;icey. Li,q/ir ,ltici,iw o p i i i i B i h , q j , i Pi.m I I I [ I / . ~ / l / l l ~ ~ J ( i l / i . I R L h e \ \ .
ohi(ll-ci. 1989. p. IX.
[ i 5 ] A fully eqiiippcd light iiiicroscopc lahoiatoq . incliidiiig iiiicromaiiipulati~~ii
;~cccssorics.can hc p u r c h a s c d ioi- under S IOOOOO. A iiiici-o\cope wlth P o l..iioid
. c:inicfii. uhtch uoi-ks \\ell for inany G U V cxpei-imenta. ciiii he ohtained
'
;it 1,nly il fI-:lctlon O f t l l l \ C O \ t .
[36] We thank Pi-ofcszor J. B. F. N . Engbert\ for thi\ moniker (namc).
1371 D Hockhtrn. E ; ~ i ~ ~ / i ~ , i i 1982.
i i . ~ r i21.
~ i ~2x33.
[;XI D. Yoge\. B. C'. R . Guillaiiiiic. _I. H. Fcndler L u i y i i i i i r 1991. 7. h23.
[39] 1. I I Clint. . S ~ i r / i uiimr -1,q,yrryurioii. Chapman 'ind Hnll. New Yurk. 1992.
p 271
I.\/~~~
[40] A c' c ' ~ \ ~ I K.
c ~ L, Fullci-, R . P. Kand. V. A . Far\egian. B / O ~ / I ~ W I1978.
17.3163
[41] , \ f ~ / ~ i i /. U[ ii dri u i i i . s i i i \ ril . \ l ~ w i h r o i i eFii\foii. (Ed.;.: S Ohki. D. DoqIc, T D.
Fl;in;ig;in. S. W Hui. E Wayhew). f'lenuni. Neu York. 1988
(421 t W. A n x k e r . A L. Undci-wood. J Phi \ . U i w i / Y S / . H S , 1463; D. F. Evan?,
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[43] D J. Mitchell. B W.Niiihaiii. .I ( h i i SOI. F ~ d r i iE, i m . 2. 1981. 77. 601
A i i c n t i o n i s also drawn t o a n imporlant \idco-eiihanced dil'fcrential tnterfci-ciicc contrast microscop!, \tudq o f the effect ofenions o n ~ e ~ i c l eJs :E.Brady.
D F. E\aiis. B Kach:ir. B W. Kinham. .I ,4111, C/iiwi, .S(IC 1984. I06. 4279.
1441 1.. A LI. Riipei-I. S. B. 1. U. Enghcrts. D Hockstra. .I. A m C 7 i r i i i . So<.1986.
INt. 3Y2l).
1451 D Papnlt;idjopolilob. <-c// Sicr/ RCIY1'978. 5. 765.
[46] f' Mucller. T. F. Chien. B. Rudy E;iJ/l/i,Y\.
.I 1983. 44. 375.
[I]F. M. Mengcr. Aiigcit,. Chc~iii.1991. 101. 1104: Aii,ycii.. C/iwi. l i i / Ed. Oi,d
[47] J Ruiz. F. M.Goni. A. Alonso. B i o i / i m i i . Bii~/~/ij,,\
Ai.rii 1988. Y37. 127.
[4X] M K Jones. C'/IVIJI. S i i . Rri.. 1992. 21. 127.
1991. 30. 1086.
Jil.\,
A i i i ~ i ~ i ~ d ~ ~ ~ [44]
i i . M . Ucno. Y. Akcchi C/inii. LPII.1991. 1801.
[2] D. D. Liisic. Li/JlJ\l7iJii,\ F r ~ i i if/ij',\ii,.\ l o ~ ~ / J / l / f c ~ l l l ~ ElSCVier.
[ S O ] Tuw short pieces on ~ c s ~ cbeh.ivior.
lc
on? a i-cviea and tlie ollier an editorial.
1993. p. 94. 118. 122. 1x9.
are \+ell aoi-th I-eading: R Lipowskq. , V m w 1991. 349. 475. .\. Maddos. ihid
[3] F. M . Mengci-. N Halachandei-. J. A i i i C'/i(wi. SOI. 1992. /14. 5862
[4] F M . Mengcr. K . Gabrielson. J h i . i % c i i i Sw. 1994, l / 6 , 1567
1993..c.j. 205.
(511 Additional c x ~ i i i p l co~f growth. fiisioii. undul;ition excretion. wounding mil
[ 5 ] G Crilc,M . Tclkes. A. Rowlond. / ' r ~ ~ ~ o p / ~ i s1932.
~ i i i i 15. 337
hcaliiis w i l l he piihlishcd shortly ( F M. Menget. S. J Lee. L i ~ i i g n i i i i r i, n press).
[6] We thank Dr. Adolph E. Smith ior pointing out to u s tlie Crilc work
2106
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