1 Institute of Molecular Biology, Department Cell Biology, Billrothstrasse 11,
A-5020 Salzburg, Austria
2 European Molecular Biology Laboratory, Structure Programme, Meyerhofstrasse 1,
D-69117 Heidelberg, Germany
* Author for correspondence (e-mail: jvsmall{at}imb.oeaw.ac.at )
Accepted 12 February 2002
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Summary |
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Key words: Actin, Cytoskeleton, Lamellipodium, Motility, Cryo-electron microscopy
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Introduction |
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When actin is polymerised in vitro, together with the Arp2/3 complex,
branched arrays can be observed (Blanchoin
et al., 2000) that arise from the integration of the Arp2/3
complex into the branch points (Pantaloni
et al., 2001
; Volkmann et al.,
2001
). The relevance of such actin branching to the in vivo
situation was suggested by studies that applied a modified method of critical
point drying for electron microscopy and immuno-electron microscopy to the
actin cytoskeleton (Svitkina et al.,
1995
; Svitkina and Borisy,
1998
; Svitkina and Borisy,
1999
). In captivating images of lamellipodia from keratocytes and
fibroblasts, branched arrays of actin filaments were identified and the Arp2/3
complex was localised at the putative branch sites. According to these
findings and those of the in vitro studies
(Mullins et al., 1998
),
Svitkina and Borisy have proposed `treadmilling of a branched actin array' to
explain lamellipodia protrusion (Svitkina
and Borisy, 1999
). However, the credibility of this model rests on
the assumption that the method adopted for electron microscopy delivers a
faithful image of actin filament networks in situ.
We have previously highlighted the particular susceptibility of actin
filament networks, as observed in lamellipodia, to distortion during various
preparative steps commonly used in electron microscopy
(Maupin-Szamier and Pollard,
1978; Small, 1981
;
Small, 1985
;
Small, 1988
;
Small et al., 1999
). And since
obvious filament branching was not previously observed in lamellipodia
prepared by negative staining methods (reviewed by
Small et al., 1999
), the
possibility of artefacts by one or another method must be seriously
considered. In this connection, we have more recently shown
(Resch et al., 2002
) that the
branching of actin filaments is induced in concentrated suspensions of pure
F-actin by the same critical point procedure as used previously on
cytoskeletons (Svitkina and Borisy,
1999
). To resolve current discrepancies over lamellipodia
organisation, including the differences observed in quick-freeze deep-etch
preparations (Heuser and Kirschner,
1980
; Hartwig and Shevlin,
1986
; Flanagan et al.,
2001
), alternative methods must be adopted for structure
analysis.
A method that obviates steps that could introduce artefacts is
cryo-electron microscopy (see also Discussion). To date, this method has been
applied mainly to viruses (Böttcher et
al., 1997; Baker et al.,
1999
), membrane proteins
(Henderson et al., 1990
;
Miyazawa et al., 1999
),
macromolecular structures (Frank et al.,
1995
; Schatz et al.,
1995
) and helical structures
(Amos, 2000
;
Beuron and Hoenger, 2001
);
examples of larger structures in vitreous ice are limited (reviewed by
McIntosh, 2001
). In the
present study we demonstrate the feasibility of applying cryo-electron
microscopy to cytoskeletons and in particular to the analysis of lamellipodia
architecture. This first report opens the way to resolving current
discrepancies over filament-filament interactions in this vital organelle of
cell motility.
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Materials and Methods |
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Video microscopy
For live imaging of cells moving over holey films, we used GFP-actin or
GFP-VASP transfected stable B16F1 clones
(Ballestrem et al., 1998;
Rottner et al., 1999
). The
holey carbon support films were UV sterilised, coated on the filmed side with
laminin (Sigma, Vienna; floating on a drop of 25 µg/ml in PBS for 1 hour)
for improved spreading of the cells, rinsed twice with PBS (150 mM NaCl, 3 mM
NaH2PO4, 8 mM Na2HPO4, pH 7.4) and
immediately transferred to dishes with cell culture medium (for details, see
Hahne et al., 2001
). B16 cells
were plated on these films and allowed to spread overnight.
Individual grids were mounted upside-down in microscopy medium (Ham's F12
medium, Sigma-Aldrich, Austria, with 10% FCS) with aluminum fluoride (50 µM
AlCl3, 30 mM NaF final concentration) in a 37°C heat controlled
chamber; the mount consisted of two pairs of sandwiched grids as lateral clips
that were fixed by grease to a coverslip and that held the grid above the
coverslip surface, with the cells down. Video microscopy was carried out as
described (Hahne et al., 2001)
using a 63x oil immersion lens and with acquisition of fluorescence and
phase contrast image pairs at intervals of 25 seconds. The individual contrast
enhanced frames from the fluorescence channel were aligned with the help of
the hole pattern seen in phase contrast and merged with a phase contrast image
previously enhanced with an edge filter.
Preparation of extracted cytoskeletons
B16F1wt mouse melanoma cells (American Type Culture Collection) were plated
on the holey carbon films and allowed to spread overnight as described above.
The formation of extensive lamellipodia was induced by the application of
aluminum fluoride 20-30 minutes prior to fixation. Cells were washed briefly
with prewarmed PBS and extracted with one of two protocols previously used as
a first step for subsequent negative staining
(Small and Sechi, 1998) or
critical point drying (Svitkina and
Borisy, 1999
). (1) Extraction/fixation in 0.25% Triton X-100 and
0.5% EM grade glutaraldehyde (GA) in cytoskeleton buffer (CB; 150 mM NaCl, 5
mM EGTA, 5 mM MgCl2, 5 mM glucose, 10 mM MES, pH 6.1) for 1-5
minutes; or (2) extraction in 1% Triton X-100 in PEM (1 mM MgCl2, 1
mM EGTA, 100 mM PIPES pH 6.9) with added 4% polyethylene glycol (PEG),
molecular mass 20 kDa. Both extraction solutions included Alexa Fluor 488
phalloidin (Molecular Probes) at a dilution of 1:300 (22 nM) as an actin
filament label for correlative light microscopy. In both cases the
cytoskeletons were post-fixed with 1% GA in CB for 20-60 minutes with the same
amount of fluorescently labelled phalloidin added.
Some samples on finder grids were transferred to a chamber filled with CB mounted on an inverted fluorescence microscope for `mapping' of cells prior to electron microscopy. Images were taken in both phase contrast and GFP fluorescence channels and merged after edge filtering of the phase contrast image. Immediately before freezing, the samples were washed with CB briefly, mounted in the freeze plunger, blotted on the front (cell) side and plunged into liquid ethane. The grids were stored in water-free liquid nitrogen until inspection.
Electron microscopy
Frozen hydrated samples were examined with a Philips CM 200 FEG at 200 kV
in TEM low-dose bright-field mode. A GATAN 626 cryostage cooled to
approximately 90 K by liquid nitrogen was used. An optimal balance between
contrast and resolution was given at a defocus of -2.5 µm, which is within
the range applied by Lepault et al. on synthetic actin filaments
(Lepault et al., 1994);
typically, holes of a diameter between around 1.5 and 3.0 µm were used for
imaging. Since the cell's outline was almost impossible to localise at low
magnification, images were taken either in proximity of the clearly visible
perinuclear area or according to coordinates established by mapping in the
fluorescence microscope.
Electron micrographs were acquired on 3.25x4 inch Kodak Electron SO-163 plate film; negatives were digitised with a Zeiss SCAI or with an Umax Astra 2400S scanner, both using a resolution of 21 µm. For improvement of image quality, a high pass filter (Adobe PhotoShop 5.5) was applied to all images, together with an appropriate adjustment of contrast.
![]() |
Results |
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|
Cryo-EM yields high resolution images of the cytoskeleton
In the cryo-electron microscope, the outline of cells was not obvious and
only correlative light and electron microscopy or changes in ice thickness
allowed their approximate location. By selecting holes in such regions where
the ice layer was sufficiently thin, clear images of actin filament arrays and
microtubules were obtained (Figs
2,
3).
Fig. 2A and B correspond to
peripheral regions showing a filopodium (A) and a lamellipodium (B), while
Fig. 2C and D show interior
lamella regions. The high incidence of filaments running closely parallel to
the cell front in Fig. 2B suggests that this region was more or less stationary at the time of fixation
(Rinnerthaler et al.,
1991).
|
|
Further images of lamellipodia are shown in
Fig. 3 and exhibit a filament
organisation more typical of protruding regions of B16 melanoma cells, as
judged by the use of negative staining on cells fixed during active protrusion
(Rottner et al., 1999)
(J.V.S., unpublished). This figure shows lamellipodia from cytoskeletons
extracted by the two alternative procedures described in Materials and Methods
and corresponding to an initial Triton/GA treatment in
Fig. 3A,C and a Triton/PEG
extraction in Fig. 3B. For the
PEG method (Svitkina and Borisy,
1999
), we consistently observed dense aggregates of material that
were not removed by relatively extensive washing in buffer and that were
concentrated, in particular, at the lamellipodium tip (presumably PEG). We
observed similar aggregates in negatively stained samples extracted with the
same protocol (J.V.S., unpublished). Nevertheless, the contrast and
organisation of filaments in lamellipodia was comparable for both extraction
methods. Fig. 3C shows a medial
region of a lamellipodium; the clarity of the filament organisation is
surprisingly close to that observed by the negative staining procedure
(Small, 1988
;
Small et al., 1999
). The actin
filament substructure is clearly resolved in these images.
Tracing of filaments in single cryo-EM images to estimate the mean length is limited by several factors including the dimension of the hole, the high filament density and low filament contrast. Nevertheless, filaments up to 0.5 µm in a 1 µm region at the front of protruding edges could be observed. In an initial analysis, we manually traced filaments and scored the length distribution. These measurements showed that approximately 30-35% of the total filament length was contributed by filaments longer than 200 nm. Further analysis using other processing procedures or stereo imaging will be required to obtain a more reliable estimate.
In this first report, we have not made direct correlations of the same regions in cells by video microscopy and cryo-EM. However, the feasibility of the approach is indicated in Fig. 4. Thus, the combined use of finder grids and the irregular hole pattern of the films allows the location of specific areas in the cryo-EM with cells labelled with a fluorescent marker. The globular structures in Fig. 4B,C are interpreted as ribosomes retained in the cytoskeleton.
|
![]() |
Discussion |
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Following extraction and fixation, the different EM procedures that have
been applied require one or more preparative steps. The critical point drying
procedure entails the most steps, including post-fixations with tannic acid
and uranyl acetate, dehydration in ethanol, critical point drying and
shadowing (Svitkina and Borisy,
1999). By contrast, negative staining is a one step drying and
contrasting procedure. Quick freeze deep etching and cryo-EM share a similar
freezing step, following blotting of excess liquid, but the deep etch method
requires further steps of ice sublimation under vacuum and contrasting by
shadowing (Heuser and Kirschner,
1980
). An important difference between the two freezing methods,
apart from the sublimation step, lies in the possibility of cryo-EM to
directly assess the local quality of freezing, namely whether the ice was
vitreous or not. With the reservations already noted on the extraction
protocol, the cryo-EM method therefore offers the least possibility of
creating artefacts in filament organisation. The disadvantage of the method
lies in the need to restrict viewing through holes in the substrate, in
addition to the expensive instrumentation and demanding imaging protocols.
The validity of analysing lamellipodia structure over holes is indicated by
the known characteristics of lamellipodia protrusion. As others have shown,
the extension of lamellipodia can readily occur above a substrate
(Izzard and Lochner, 1980). In
the case of B16 melanoma cells, advancing lamellipodia are typically 2-5 µm
across and protrusion on laminin is supported by adhesion at the lamellipodium
base by focal complexes (Rottner et al.,
1999
). It can therefore be assumed that lamellipodia extending
over holes of 2-3 µm in diameter have a structure typical of neighbouring
regions extending over substrate. The present findings thus pave the way for a
critical analysis of lamellipodium architecture. By pursuing this approach in
combination with 3D imaging (Grimm et al.,
1997
) it should be possible to reach a consensus on the extent of
actin filament branching in lamellipodia. Likewise, future prospects include a
more precise localisation of the many lamellipodia-associated proteins
(reviewed by Small et al.,
2002
) to shed further light on the structure-function
relationships underlying actin-based protrusion in cell motility.
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Acknowledgments |
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