Traffic 2000 1: 545–552
Munksgaard International Publishers
Toolbox
The Production of ‘Cell Cortices’ for Light and
Electron Microscopy
Department of Cell Biology, Washington University School
of Medicine, St. Louis, MO, USA
[email protected]
the inner surface of the plasma membrane has evolved over
the years (10 – 12), and to describe the procedures we currently consider to be optimal for attaching cells to polylysinecoated coverslips, for ‘unroofing’ them, and for imaging them
in the electron microscope.
Received 17 March 2000, accepted for publication 11
April 2000
Sticking Cells Down: Choice of the ‘Glue’
John Heuser
A Brief History of the Technique
The inner surface of the cell membrane offers a wealth of
structural information that is vital for understanding the behavior of the cell cortex in particular, and the whole cell in
general. A quarter of a century ago, Mazia et al. (1), Clarke et
al. (2), Vacquier (3), and their colleagues at Berkeley introduced a powerful method for viewing this surface by light
and electron microscopy, which, in principal, remains unchanged to this day. They were the first to realize that
polylysine (and other cationic polypeptides) adsorb strongly
to various solid surfaces creating a high density of free
cationic sites that strongly ‘glue’ cells down through electrostatic interactions with the negative charges that predominate on the cell surface. In their original report (1), these
authors noted that cells thus attached to polylysine surfaces
Polylysine remains the ‘glue’ of choice in most circumstances; however, alternatives have come and gone over the
years. Thus, Buechi and Bachi (13), virologists in Zurich
wishing to see the inner aspect of the plasma membrane at
sites of Sendai virus insertion and budding, described a
procedure for cationizing glass with an amino-silane compound dissolved in acetone [see also (14)]. After covalently
attaching this compound to the glass via its silane groups,
these workers then exposed the glass to glutaraldehyde to
convert the free amino groups into reactive aldehydes. This
‘glued’ cells to the glass covalently by the formation of
aldehyde bonds, and was quite successful at creating a ‘grip’
that resisted cell rupture and subsequent preparations for
electron microscopy (EM). Their amino-silane technique has
been used occasionally since then (14) but has proved to be
‘remain alive, but may flatten or spread themselves to a
degree that is not normal but may be desirable for obser vational purposes.’
More importantly, they introduced the technique that will be
elaborated and brought up-to-date in the present ‘Toolbox’
with the simple statement (1):
‘One interesting application of the method is in the obser vation of the inner face of the cell surface. The cells are
attached to the polylysine surface and subjected to shear
in a medium in which the cytoplasm will disperse when
the cell membrane is torn. The body of the cell is sheared
away but the area of the cell surface which is glued by the
polylysine remains attached, inner face up.’ (Figure 1).
Soon after the introduction of this technique, Jacobson and
colleagues adapted it for biochemistry in a highly useful
manner, by developing a technique for coating glass beads
with polylysine, attaching cells to the beads and then rupturing them, and thereby collecting sufficient quantities of
plasma membranes for interesting biochemical analyses (4 –
9). Unfortunately, this informative biochemical approach has
fallen into relative disuse. In any case, the goal of the present
report is to describe how the ‘Mazia’ technique for imaging
Figure 1: Vacquier’s original image of the cortex of a sea
urchin egg attached to polylysine-coated glass and ‘unroofed’ by a squirt of buffer. This swept away the cytoplasm of
the egg and left only the attached cortical granules that were
attached to the inside of the plasma membrane and in position
for exocytosis. Reprinted with permission from reference (3).
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Figure 2: Anaglyph stereo view of our apparatus for ‘unroofing’ cells by exposing them to a gentle ‘blast’ from an ultrasonic
probe immersed in a dish of buffer. For successful application of this technique, close visual monitoring must be established. This
we accomplish by viewing the probe tip and sample through a high-magnification dissecting microscope projected through a mirror
angled at 45°. [For discussion on the preparation of anaglyph stereo images see (41)]1.
too tedious for routine use and, in our hands, offers no
special advantages over the original polylysine technique.
Nonetheless, when aldehyde fixation rather than electrostatic bonding of cells to glass is desirable, an adsorbed film
of polylysine can also be reacted with glutaraldehyde without
displacing it from the glass. This converts all the polylysine’s
free epsilon-amino groups to free aldehyde groups and renders the glass electroneutral, just like a glutaraldehyde-fixed
amino-silane surface. In our experience, cells survive the
attachment to such ‘aldehyde monolayers’ just as well as to
poly-cationized surfaces, and sometimes survive and adhere
even better!
Types of Cells that are Suitable, and a
Precautionary Note
Until recently, this technique has generally been used with
cell suspensions that are adsorbed acutely to the polylysine
and ‘unroofed’ after just a minute or two of adhesion. Thus,
it has been ideal for free-living cells like amoebae (2,15) or
naturally suspended cells like erythrocytes (16) and leukocytes (10). Likewise, it has also been used with tissue-cultured cells that have been acutely resuspended with
trypsin-EDTA, or even with whole tissues that have been
dissociated into individual cells with collagenases. In any
1
Editorial footnote: Dual red/blue glasses are required to see
anaglyphs in 3-D, and are available in Traffic volume 1, issue 1,
or can be purchased for a nominal fee at http://
stereoscopy.com/3d-images/
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case, it is important to realize that regardless of the source of
cell-type used, the final cell suspension that is about to be
glued down onto polylysine must always be washed carefully
by repeated gentle centrifugation in protein-free saline. Furthermore, great care must be taken to avoid any cell rupture
during these washes, or else cytoplasmic proteins will be
liberated. The problem here is that any free protein outside
the cells will adsorb to the polylysine-coated surface and
promptly obliterate its stickiness, before the cells have had a
chance to attach.
Thus, this technique stands in stark contrast to the commonly used technique of pretreating glass or plastic with
polylysine before tissue culturing. In this situation, full,
serum-containing medium is always applied to the polylysine
before or during the introduction of the cells and the high
concentration of proteins in the medium – molecules such
as fibrin, etc. – adhere to the polylysine and form a relatively
‘natural’ surface for the cells to grow on. In contrast, most
tissue-cultured cells (and particularly very motile ones) do not
even survive very long on ‘bare’ polylysine; the grip is just
too tight and they tear themselves apart. The important point
to stress here is the following: if adhesion of a cell suspension to polylysine fails, it is generally because (1) cell damage
has occurred during the wash steps, (2) the cells are intrinsically too fragile, or (3) their exposure to the glass has gone
on too long and they have begun to break open ‘spontaneously’. We find that even after cells have been adsorbed to
polylysine-treated glass, the release of proteins from a few
broken ones will dislodge them all.
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Production of Cell Cortices
‘Unroofing’ Polylysine-Attached Cells
The original approach of shearing open strongly adherant
cells with a simple ‘squirt’ of buffer (1) has remained standard (11), but many variations have also been tried. Thus, the
‘squirt’ may come from a Pasteur pipette or from a needleand-syringe; or as we prefer, it may come from the intense
displacement of liquid caused by the standard type of ‘horn’type ultrasonic probe (17,18). Our current protocol is to use
one of the one-eighth inch diameter ‘microprobes’ originally
designed to sonicate small samples in Eppendorf tubes,
driven by a very gentle 10–25 W power supply running at
only 10–20% of maximum power. This is dipped into 10
ml of ‘breakage buffer’ (basically, any K/Mg based isotonic
buffer) in a 60 mm Petri dish, such that when power is
applied, a jet of liquid and tiny cavatation-bubbles emerge
straight down from the probe tip. A coverslip of polylysineadherent cells is inserted into this dish of buffer at a 45°
angle, positioned about 1/8th inch below the probe tip, and a
momentary blast is delivered by a foot-control on the ultrasonic power supply.
This is all done under strict visual control, with a bright 80×
dissecting microscope viewing the sample from the side
through a 45° mirror, since in that way it is easy to see the
slight turbidity on the glass created by adsorbed cells and
easy to see when they are partially ‘blown away’ (Figure 2).
It is vital to stress that such partial cell removal is the desired
goal, since only then can one be certain that some cells will
be just barely unroofed and the maximum amount of cortical
structure will remain on their inner surfaces! Direct visualization of this ‘unroofing’ step also shows when the jet of liquid
has ‘misfired’ or has otherwise been inadequate for un-
roofing the cells in question, or when it has been too harsh
and has totally denuded the coverslip. (In lieu of such a
setup, one can simply ‘unroof’ several coverslips with different times or power settings of sonication and look for the
best result. Cell cortices remain visible in standard tissue-culture type inverted light microscopes, albeit as nothing more
than faint dark rings or ‘smudges’ on the glass where the
cells used to be.)
‘Squashing’ Cells as an Alternative to
‘Unroofing’ them by Fluid Shear
The major alternative to ‘unroofing’ cells by a shearing fluidflow has been to ‘squash’ them gently between two sticky
coverslips (19,20) or between a sticky coverslip and an EM
grid (21). In our hands, this works quite well for cells that are
so tough that they refuse to yield to a shearing force, or that
adhere so weakly to polylysine that they are always completely detached without leaving behind their ventral surfaces. It is also useful for cells that are grown on
non-adherent surfaces such as porous filters, as epithelial
cells often are, especially when an EM view of the cell apex
is desired (19 – 21). By squashing cells between two coverslips or any two opposed surfaces, they can’t get away!
Additionally, their organelle ‘guts’ often are spilled out onto
the opposed surfaces during the squash, and these are often
informative, in and of themselves. Finally, such ‘squashing’
of course yields on the upper coverslip a sample consisting
primarily of cell apices, whose structure can then be compared with the ventral surfaces normally obtained by the
standard ‘unroofing’ procedure. The only problem with this
‘sandwich’ technique is that it generally yields much
‘messier’ preps on polylysine-coated glass surfaces. This is
Figure 3: Nermut’s image of the inner surface of a cultured hepatocyte
‘unroofed’ by a squirt of buffer and
then prepared for high-resolution
TEM by fixation and critical-point
drying. This clearly displayed the polygonal clathrin lattices that abound on
this actively endocytic membrane surface. Reprinted with permission from
reference (22).
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Heuser
because it takes some time to separate the two coverslips
after the squash, thus delaying the washing and fixation
steps that are normally carried out immediately after
‘unroofing’.
Note of an Unexplained ‘Artifact’ that Plagues
this Technique!
Jumping ahead to the final visualization and interpretation
steps of the techniques outlined here, we should note immediately that cells acutely attached to polylysine (or to other
cationized surfaces or even to aldehyde-coated surfaces) in
the absence of any exogenous proteins, display one huge
artifact in their plasmalemmae! Their adhesion must be so
tight that their plasma membranes become ‘embossed’ with
an odd texture that is visible at high magnification in the EM.
This we don’t completely understand. Perhaps the texturing
is a reflection of inhomogenities in the distribution of cations
on the glass (or anions in the cell membrane), or perhaps it is
actually proteins (maybe even the cells’ own membrane
proteins) trapped between the membrane and the glass. In
any case, such ‘embossing’ is clearly an artifact, since it is
entirely absent from regions of the plasma membrane that
naturally curve inwards, such as nascent pinosomes or the
bases of ruffles. Furthermore, it is entirely absent from cells
that are ‘unroofed’ in the absence of polylysine, as will be
described next.
‘Unroofing’ Cells on their Original
Growth-Surfaces
Perhaps the single most useful advance made over the
original Mazia technique has been the realization that tissuecultured cells need not be removed from their primary culture environments, resuspended and then acutely plated on
polylysine: instead they can be grown directly on acidcleaned glass and ‘unroofed’ while still on the original glass
(22 – 25). Often, the cells’ grip to the glass is sufficient for at
least some of their ventral surfaces to hang on throughout
the ‘unroofing’ procedure. Importantly, these cells show
none of the plasmalemmal embossing artifact described
above. For ‘unroofing’ such long-term cultures, we have
found that we can vastly increase the yield of properly
exposed ventral cell bottoms by an elaboration of the original
procedure. Specifically, we expose the cultures very briefly
to a relatively low molecular weight polylysine, just long
enough to allow it to diffuse under the outer edges of the
cells and begin to glue their perimeters down onto the glass.
This region again acquires the ‘embossing’ described above,
and thus is ruined for high-resolution EM; nevertheless, it
serves to hold down the more central regions of the cell
cortex, which are not so embossed.
To make this ‘edge-gluing’ trick work properly, the cell cultures must first be washed free of all protein, again by
several rinses in warm PBS to remove their original culture
medium. Only then can they be exposed to polylysine without fear of creating a precipitate of protein. To achieve the
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Figure 4: Anaglyph stereo-view of the inner surface of a
PC12 neuroendocrine cell. It was exposed by the ‘unroofing’
procedure described herein, displaying a number of chromaffin
granules (secretory vesicles attached to the plasma membrane
by long ‘threads’ and/or by actin filaments in the cortical cytoskeleton).
actual ‘edge-gluing’ effect, we generally use 0.1 mg/ml of
polylysine dissolved in PBS, using the 30 – 60 kDa oligomers
of polylysine from Sigma (St Louis, MO). Coverslips are
swirled in this solution very briefly – for only 15 s – before
being rinsed to arrest any further penetration of polylysine
under them and to remove any excess polylysine that might
contaminate their inner surfaces once they are ‘unroofed’.
Still, even this brief exposure to a relatively dilute polylysine
solution creates a severe precipitate on the outsides of cells,
which is readily visible on the cells that happen not to be
‘unroofed’ in the next step. One must hope that this precipitation doesn’t have enough time to seriously alter cell
physiology.
One further aid to successful unroofing of cells that reside on
their original glass coverslips is to swell them gently in the
last moments before sonication. For this, we use a one-third
osmotic strength solution. Finally, since the interior of the
cell cortex is about to be exposed, it is only logical to
immediately precede the ‘unroofing’ procedure with an appropriate change in salts from the usual Na + /Ca2 + base of
the whole cells’ external world, to the K + /Mg2 + base of
their internal world. Putting all these considerations together,
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Production of Cell Cortices
we unroof cells grown in long-term cultures on their own
coverslips by the following protocol: 1) transfer them from
full medium in the incubator to three washes in 37°C PBS
over 15 min; 2) then pass them through 15 s in 0.1 mg/ml
‘mid’-molecular weight polylysine in Ca-free PBS; 3) then
immediately wash them in three changes over 30 s in onethird strength K+/Mg2 + buffer; and 4) finally transfer them to
the sonication bath containing full-strength K+/Mg2 + buffer
(9 sucrose to stabilize internal organelles) and ‘unroof’ them
by an approximately half second ultrasonic ‘burst’.
to preserve the architecture of the cortex in as natural a state
as possible, so chemical fixation or ‘quick-freezing’ will be
mandated as soon after the ‘unroofing’ as possible. When
fixatives are used here, the choice is straightforward: glutaraldehyde for the best structural preservation or formaldehyde for the best antigen preservation, but in either case, in
the same K + /Mg2 + buffer as was used for the unroofing.
It is worth noting that ‘unroofing’ cells grown in culture of
course yields only their original, ventral surfaces, whereas
unroofing cells acutely plated on polylysine gives a random
sampling of their ventral and apical surfaces (presuming that
during their resuspension at 37°C, such regional differentiations were not lost).
Next comes the question of how to get such ‘unroofed’ cell
preparations on polylysine-treated glass into the electron
microscope. Again, Mazia et al. (1), Clarke et al. (2) and
Vacquier (3), in their original studies, used standard OsO4
postfixation, ethanol dehydration, and critical-point drying before platinum-coating and imaging of their preparations with
high-resolution SEM (Figure 1). So also did Nermut (16) and
others in their extensive use of this technique [see also
(5,15,17)] (c.f. Figure 3). For decades we have advocated
freeze-drying and TEM over critical-point drying and SEM,
partly because it obviates the potentially damaging effects of
osmification and dehydration, and partly because TEM is so
much easier to do than high-resolution SEM (26 – 29).
Preserving ‘Unroofed’ Cells for Light and
Electron Microscopy
Having now successfully ‘unroofed’ cells by one means or
another, the next question is how to prepare them for microscopy. We have found that their exposed ventral membranes will ‘survive’ in K/Mg buffer for 10 min before they
become severely vesiculated. During this time, the actin
filaments and microtubules that initially cling to them progressively depolarize, and the clathrin-coated pits on them
round up, as if ‘in preparation’ for pinching off. Remarkably,
however, their caveolae remain totally unperturbed and do
not change their curvature. If desired, experiments like
adding cytosol, etc., can be carried out on the ‘unroofed’
cells during these 10 min, or they can be briefly decorated
with antibodies. More commonly, however, the aim will be
Final Preparative Steps for Getting ‘Unroofed’
Cells into the EM
In the end, the choice between critical-point drying and
freeze-drying is largely a matter of personal preference, and
becomes a decision based largely on whether the proper
technology and expertise for decent freezing is locally available, or not. ‘Quality’ freeze-drying cannot be carried out if
the freezing itself is not optimal; here we believe that only
our liquid helium-cooled ‘Cryopress’ achieves truly optimal
freezing (30). Earlier studies of plasmalemmal lawns suffered
technically exactly at this point. Buechi and Bachi (13,31), for
Figure 5: Anaglyph stereo-view of the
inner surface of an ‘unroofed’ CHO
cell subjected to indirect EM immunocytochemistry with anti-caveolin antibodies and 15 nm gold-tagged
secondaries. Dome-shaped caveolae
are clearly and specifically labeled with
gold (yellow dots), in contrast to the
unlabelled actin filaments and the one
clathrin-coated pit in the field.
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Heuser
Figure 6: HRP-labeled endosomes
from
a
Dictyostelium
amoeba
‘squashed’
on
polylysine-coated
glass. (Note the abundance of released
ribosomes stuck to the glass in the
background.) The DAB product of the
HRP reaction looks white in such contrast-reversed anaglyph stereos.
example, froze their preparations by plunging them into liquid
nitrogen – a clearly inadequate approach. Ohno achieved
somewhat better freezing by plunging his preparations into a
mixture of pentane and propane cooled with liquid nitrogen
(14). Following our earlier advice that methanol acts as a
cryoprotectant, which will evaporate during freeze-drying
(27), he also took the further step of washing his preps in
10% methanol before freezing. In any case, no cryo-‘plunging’ technique achieves the rates of cooling and minimizes
ice crystal formation as much as does ‘slam-freezing’ against
an ultra-cold metal block; that is why we advocate our original freezing procedures (30). The Cryopress cushions the
sample in such a way that controlled ‘slamming’ of glass
coverslips onto ultracold metal does not risk breaking the
glass.
Quality freeze-drying also depends on the procedures followed after freezing. In general, this freeze-drying is done in
a vacuum-evaporator, so that a metal replica can be applied
immediately thereafter. But this requires transfer of the
frozen sample onto a ‘cryo-stage’ inside the vacuum-evaporator where it can be kept frozen, and very few investigators
have worried about the problems inherent in this transfer:
namely, the transient warming of the sample and the accumulation of condensed atmospheric moisture or ‘hoarfrost’
on it. We avoid these ‘perils’ by capping the sample while it
is still immersed in liquid nitrogen, and by not removing its
cap until transfer into the evaporator is complete and the
sample is firmly stabilized at −150° within a fully-evacuated
chamber. Furthermore, few practitioners of freeze-drying
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have worried about the hazards of leaving samples too long
in the vacuum after freeze-drying. Buechi and Bachi (13) left
their samples to freeze-dry for 90 min at − 40° and Ohno
(14) freeze-dried for 60 min at − 90°C, while we find that
thin films of water on glass coverslips are completely freezedried in only 15 min at − 100°C (18). So we get the freezedrying over with fast, and we watch its completion with a
high-power dissecting microscope mounted in the evaporator; and, just as soon as possible, we create our platinum
replica of the glass surface. All of these precautions serve to
sustain the maximal surface architecture of our ‘unroofed’
cells and explain why they display such dramatic 3-D topology (Figure 4).
A ‘Shortcut’ Especially Useful for
Immunocytochemistry of ‘Unroofed’ Cells
We should add here that a ‘shortcut’ used by many labs to
get such ‘unroofed cell’ preparations – or ‘plasmalemmal
lawns’ as they are often called (32,33) – into the electron
microscope without any replication at all is simply to briefly
stain and air-dry them. This has been carried out most successfully in a number of immunocytochemical studies,
where gold-labeled secondary antibodies showed up very
nicely due to the thinness of the sample and the faintness of
its staining (32 – 35). Unfortunately, the quality of structural
preservation is rather compromised by the air-drying used in
these studies, so proper identification of structural details is
often rendered somewhat ambiguous and nothing remains
of the cells’ natural 3-D topology.
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Production of Cell Cortices
We strongly advocate such use of EM immunocytochemistry
on ‘unroofed’ cells, particularly because such preparations
offer perfect exposure to antibodies without the need for any
detergent or formaldehyde fixation. However, we would suggest that it is perfectly possible to combine such EM immunocytochemistry with proper freeze-drying and platinum
replication of ‘unroofed’ cells, and thus preserve their surface
architecture as well. The gold particles that mark the secondary antibodies are readily visible underneath a platinum
replica. This is equally true when indirect EM immunocytochemistry is performed on the residue of proteins that cling
to the undersides of platinum replicas made from whole
tissues (36).
they are intrinsically so thin that excellent fluorescence microscopy can be done on them, without the need for any
form of ‘confocal’ imaging (38).
Finally, it is worth remembering that such substrate-attached
cell cortices are not only great for all sorts of microscopy, but
are also excellent preparations for a variety of in vitro reconstitution assays, providing ideal substrates for studying dynamic membrane processes such as actin polymerization,
exocytosis (39), and endocytosis (20,32 – 35,40).
References
1.
Final ‘Secrets’ for Successful Gold and
HRP-Labeling of Freeze-Dried Cell Cortices
The only problem with EM immunocytochemistry of ‘unroofed’ cells on polylysine-coated glass is that the gold particles tend to get ‘eaten away’ when the platinum replica is
floated off the glass by angling it into a dish of hydrofluoric
acid, as is standardly done. To overcome this problem, we
have introduced one final ‘wrinkle’ in the original technique of
Mazia et al. (1). Specifically, we pre-coat the glass coverslips
on which we grow cells (or to which we acutely glue-down
suspended cells) with a thin layer of carbon. Having done so,
the final platinum replica separates in hydrofluoric acid along
the carbon/glass interface. Hence, the gold antibodies – now
‘sandwiched’ between the platinum replica above and the
carbon beneath – remain totally unscathed (Figure 5). In fact,
the entire ventral cortex of the ‘unroofed’ cells remains
within this ‘sandwich’ as well.
2.
3.
4.
5.
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Were this cortex any thicker, it might obscure detail or add
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before or after sonication and, more importantly, it also
allows oxidized DAB to be visualized within an actual biological sample as well (Figure 6). Thus, HRP extracellular-tracers
and HRP-labeled antibodies can be visualized in such freezedried and platinum-replicated samples, just as well as gold.
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Conclusion: The General Utility of ‘Unroofed’
Cell-Cortex Preparations in Cell Biology
In the world of light microscopy as well as that of TEM and
SEM, ‘unroofed’ cell cortices can also be highly informative,
especially when imaged by high-contrast techniques like interference-reflection microscopy (15,24,37) or video-enhanced differential interference microscopy (39). Moreover,
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