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Cell Motility and the Cytoskeleton 35345-357 (1996)
Effects of Electroporation on the Tubulin
Cytoskeleton and Directed Migration of
Corneal Fibroblasts Cultured Within
Collagen Matrices
Damien G. Harkin and Elizabeth D. Hay
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts
Electroporation provides a useful method for loading fibroblasts with fluorescent
probes for the cytoskeleton, but the possible deleterious effects of this loading
technique on cell motility are unknown. We have used conventional and confocal
microscopy of living cells and immunohistochemistry to examine the migration and
cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured
within collagen gels. Fibroblasts cultured in collagen ( 1 mg/ml) are successfully
electroloaded (0.5-1.0 kVcm-'/960 pF in DMEM/F12/20 mM Hepes, pH 7.2)
with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one
direction. The lack of directed movement is due to depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by
millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (510
pM Ca2+)maintain their cytoskeleton and continue to migrate, when returned to
culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin
and monitored the behavior of the labeled actin cortex by confocal microscopy.
During migration phalloidin-actin accumulates near the base of pseudopodia and
at the rear of the cell where it is subsequently left behind. We conclude that
electroporation is a valuable technique for loading fibroblasts to study migration
within ECM, provided that the conditions used support stability of the tubulin
cytoskeleton. 0 1996 Wiley-Liss, Inc.
Key words: cell motility, extracellular matrix, microtubules, vimentin, F-actin, live confocal microscopy
INTRODUCTION
bedded in collagen gels are difficult to load with probes
for the cytoskeleton by using traditional methods such as
The migratory behavior of fibroblasts, as for that of
microinjection. We therefore chose, in the present study,
many cell types, is readily studied using cells attached to
to investigate the use of electroporation as a method for
planar substrata [Abercrombie et al., 1970a, 1970b,
loading fibroblasts suspended in collagen gels with mac1972; Vasiliev et al., 1970; Heath, 1983; Wang, 1985;
romolecules.
Theriot and Mitchison, 1992; Jones et al., 19931, but this
Electroporation (electro-permeabilization, electromethod invariably introduces artifacts associated with
injection) is widely used as a method for transfecting
the cells having an upper free surface [Bard and Hay,
19751. Reconstituted collagen matrices [Elsdale and
Bard, 19721 therefore provide a valuable means for Received May 20, 1996; accepted August 9, 1996.
studying fibroblast migration within a 3-D substrate of Address reprint requests to Damien G. Harkin, Ph.D., Department of
extracellular matrix (ECM) components similar to that Cell Biology, Harvard Medical School, 220 Longwood Avenue, Bosfound in vivo [Hay, 1985, 19911. However, cells em- ton, MA 021155729,
0 1996 Wiley-Liss, Ine.
346
Harkin and Hay
cells with nucleic acid, but this method has also been
used to load cells with a variety of other molecules including phalloidin [Hashimoto et al., 1989; Glogauer
and McCulloch, 19921 and antibodies against the cytoskeleton [Wilson et al., 1991al. Although the precise
mechanism of cell electro-permeabilizationis debatable,
it is apparent that brief electrical pulses temporarily open
plasma membranes to molecules which are normally too
large or hydrophilic to enter cells by diffusion [Tsong,
1991; Neil and Zimmermann, 1993; Weaver, 1994,
19951. However, the conditions required for electroporation of cell membranes are often associated with a decrease in cell viability. Moreover, methods commonly
used to assess the health of cells following electroporation, such as dye exclusion [Liang et al., 1988; Wilson et
al., 1991b;Glogauer and McCulloch, 1992; Baum et al.,
19941 or proliferation in culture [Tatsuka et al., 1988;
Lukas et al., 1994; Wolf et al., 19943, provide a poor
indication of the motile capacity of cells immediately
following loading. The suitability of electroporation as a
tool for studying cell migration is therefore unclear.
In the present article, we examine the effects of
electroporation on the permeability, migration and cytoskeleton of chick embryo corneal fibroblasts cultured
within hydrated collagen gels. Corneal fibroblasts (keratocytes), like other types of cranio-facial mesenchyme,
are derived from motile neural crest cells, and continue
to migrate in the chick until 12 days of embryonic development [Hay and Revel, 19691. We establish optimal
parameters for electroporating motile corneal fibroblasts
by loading various sizes of fluorescent dextran and by
measuring retention of cytoplasmic esterase activity with
fluorescein diacetate. The motile behavior of fibroblasts
following loading is monitored by both conventional and
confocal time-lapse microscopy. We develop an electroporation buffer which preserves the normal migratory
behavior of fibroblasts in collagen gels and facilitates
stability of the tubulin cytoskeleton as demonstrated by
immunohistochemistry. The effectiveness of this approach for studying the cytoskeleton, is demonstrated by
monitoring the F-actin cortex of living cells electroloaded with rhodamine phalloidin.
MATERIALS AND METHODS
Isolation of Corneal Stroma
Six-day-old White Leghorn chick embryos (Spafas, Norwich, CT) were dissected in Hanks' balanced
salt solution containing 20 mM Hepes (HBSS-Hepes, pH
7.2) and their corneas removed using a 2 mm trephine
blade (Storz Instrument Co., St. Louis, MO). Excised
corneas were digested in 0.25% trypsidHBSS-Hepes for
15 min at 37°C to remove epithelia, then placed in
DMEM/Fl2 medium supplemented with 5 % fetal bovine
serum (EBS). Corneal stromas were then cut into several
pieces and suspended in collagen gels.
Preparation of Collagen Gels
Type I collagen from rat tail (RTC) was obtained
from Collaborative Biomedical Products (Bedford, MA)
as a solution in 0.02 N acetic acid, and dialyzed at 4°C
against a 10% solution of F12 medium (without bicarbonate; pH 4.0). The concentration of dialyzed RTC was
determined by Sirius Red precipitation [Marotta and
Martino, 19851and adjusted to 3.0 mg/ml with 10% F12
medium. A stock solution of polymerizing collagen was
prepared on ice by combining dialyzed RTC, with a 10
times concentrated solution of F12 medium (without bicarbonate), 140 mM sodium bicarbonate and FBS, in the
ratio of 7:l:l:l [Elsdale and Bard, 19721. The collagen
solution was further diluted to 1.0 mg/ml with cold
DMEM/F12/5% FBS and immediately dispensed as 50
pl aliquots into rings of Whatman No. 1 grade filter
paper (inner diameter, 6 mm; outer diameter, 10 mm)
placed in a plastic petri dish. One piece of corneal stroma
was added to each ring of filter paper and set in place by
polymerizing the collagen at 37°C. The corneal stromas
in collagen were then cultured for 18-24 h in DMEM/
F12 supplemented with 5 % FBS, 0.4 mM L-glutamine,
50 pg/ml ascorbic acid, 2.5 p,g/ml fungizone, 5.0 pglml
gentamicin, and 1% ITS + medium supplement (Collaborative Biomedical Products).
Preparation of Macromolecules
Tetramethyl rhodamine isothiocyanate (TRITC)
dextrans (4 kDa and 76 kDa) and fluorescein isothiocyanate (FITC) dextrans (4, 40, 70, and 150 kDa) were
obtained commercially (Sigma Chemical Co., St. Louis,
MO) and dialyzed in PBS to remove trace amounts of
unbound fluorophore. Fluorescein isothiocyanate (FITC;
Sigma) was conjugated to chicken IgG (Sigma) as previously described [F'ryzwansky, 19821. All preparations
of fluorescent macromolecules were equilibrated with
electroporation buffer by dialysis, sterilized by filtration,
and stored at 4°C. Rhodamine phalloidin (RPh; Molecular Probes, Eugene, OR) was dissolved in methanol at
a concentration of 6.6 pM and stored at -20°C. For
electroporation, 500 p1 of RPh stock was dried, dissolved in 1 ml of electroporationbuffer, and sterilized by
filtration prior to use.
Electroporation Buffers
Three electroporation buffers were tested for effects on the motility and/or cytoskeleton of fibroblasts
cultured in collagen gels: (1) serum-free DMEM/F12
without phenol red (Gibco BRL Cat. No. 21041-025;
Life Technologies, Grand Island, NY), buffered with 20
mM Hepes, pH 7.2 (DFH); (2) cytoskeleton stabilizing
Effects of Electroporation
buffer (CSB) as used for immunostaining (refer below);
and (3) a buffer referred to as “intracellular” electroporation buffer or ICEB, which we designed to mimic the
ionic composition of intracellular fluid (125 mM KOH,
4 mM NaOH, 73 mM Pipes free acid, 34 mM myoinositol, 10 mM NaHC03, 5 mM K,HPO,, 5 mM
KH,PO,, 5 mM D-glucose, 4 mM MgCl,, 1 mM
MgSO,, 1-10 p M CaCl,, pH 7.0).
347
Video Microscopy
A preliminary assessment of the effects of electroporation on the speed and direction of fibroblast migration were made using conventional light microscopy.
Collagen gels containing corneal fibroblasts were placed
in a 35 mm petri dish which had been modified for microscopy by cutting a 16 mm diameter hole in the bottom
and attaching a 22 mm2 no. 0 glass coverslip with silicon
glue.
A 12 mm diameter round coverslip was then placed
Electroporation
on top of each gel and allowed to settle before adding 2
Collagen gels containing explants of corneal ml phenol red-free culture medium buffered with 20 mM
stroma were removed from culture and washed three Hepes (pH 7.2) followed by 2 ml silicon oil. Cultures
times for 10 min each in 10 ml serum-free DMEM/ were maintained at 37”C, covered with 5% CO, in air
F12/20 mM Hepes (pH 7.2), then rinsed for an additional and mounted on a Zeiss IM-35 inverted microscope us5 rnin in three changes of electroporation buffer. Washed ing an LU-CB-1 tissue culture chamber and TC- 102 temgels were then incubated for 15 rnin at room temperature perature controller (Medical Systems Corp., Greenvale,
(25°C) in electroporation cuvettes (0.4 cm electrode gap, NY). Time-lapse recordings of cell migration were made
Bio-Rad Laboratories, Hercules, CA) containing 750 under phase (one every min for up to 2 h) using a Zeiss
p1 of electroporation buffer with macromolecules. Cul- 63 X A.25 n.a. Neofluar oil immersion lens and DAGEtures were electroporated at room temperature using a MTI NC-66 X video camera (DAGE-MTI Inc., Michisingle exponential pulse delivered from a Bio-Rad Gene gan City, IN) interfaced with Metamorph image analysis
Pulser transfection apparatus (Bio-Rad). Various combi- software (Universal Imaging, West Chester, PA). Loadnations of field strength (500-1,000 Vcm-’) and capac- ing of cells was confirmed at the end of each recording
itance (125-960 FF) were tested for their effects on the by switching to epifluorescence. Distances migrated by
uptake of macromolecules and cell viability. Immedi- cells were defined by the location of nuclei and deterately following electroporation, gels were removed from mined using the measure function of the Metamorph procuvettes and incubated for 10 min in fresh electropora- gram, calibrated with a micrometer slide.
tion buffer at 37°C to facilitate resealing of cell membranes, then returned to room temperature and washed Live Confocal Microscopy
for 1 h in several changes of DMEM/Fl2/Hepes (pH 7.2)
Cultures of corneal fibroblasts in collagen were
without phenol red, to remove excess macromolecules.
prepared as above for video microscopy, then mounted
Cell Loading and Viability Assay
on a Zeiss LSM confocal microscope equipped with enCollagen gels containing electroporated fibroblasts vironmental chamber (37”C, 5% CO,). Paired pseudowere placed on a glass slide in phenol red-free culture Nomarski and confocal fluorescence images (average of
medium and covered with a no. 0 glass coverslip. Slides 8 line scans) were obtained using a 40 x /1.3 n.a. Neowere mounted on a Zeiss (Thornwood, NY) Axioskop fluar lens and 488 nm or 568 nm krypton-argon laser line
fluorescence microscope equipped with 200 watt mer- attenuated to 1%. The pinhole aperture for confocal flucury lamp and examined using a 63 x /1.25 n.a. Plan- orescence was adjusted to produce a ‘Z’ resolution of
Neofluar oil immersion lens. At least 100 cells were approximately 1 pm. Images were collected manually at
examined per gel and the percentage of fluorescent 15-min intervals for up to 2 h.
loaded cells calculated. The cytoplasmic distribution of
loaded macromolecules was confirmed by live confocal lmmunostaining
microscopy.
Collagen gels containing corneal fibroblasts were
Cell viability was assessed by measuring the pro- rinsed briefly in cytoskeleton stabilizing buffer (CSB;
portion of cells with sufficient cytosolic esterase activity 100 mM Pipes free acid, 160 mM NaOH, 20 mM myoto convert fluorescein diacetate to its fluorescent prod- inositol, 5 mM MgCl,, 5 mM EGTA, pH 6.8), extracted
uct. Collagen gels containing fibroblasts were incubated with 1% Triton X-100/4%polyethylene glycol-8000 in
for 10 min at 37°C in a 1 FM solution of fluorescein CSB (15 rnin), fixed with 2.0% (w/v) paraformaldehyde
diacetate in phenol red free culture medium, then washed in CSB (15 min), washed in PBS (two times for 15 min),
briefly in fresh medium before being examined as above. then incubated overnight at 4°C in 0.15 M Tris buffer
Simultaneous measurements of loading and viability containing 50 mM NH,Cl and 1% normal goat serum
were achieved by using cells electroporated in the pres- (NGS). Gels were then immunostained for tubulin or
vimentin by incubation for 1 h at 37°C in 250 (1.1 of E7
ence of TRITC-dextran (4m a ) .
348
Harkin and Hay
hybridoma supernatant containing IgG 1 raised against
drosophila p tubulin, or H5 hybridoma supernatant containing IgGl raised against chick vimentin (both obtained from Developmental Studies Hybridoma Bank,
Iowa City, IA; refer to "Acknowledgments"), followed
by 1 h at 37°C in 250 pl of 1:lOO affinity purified, goat
anti-mouse rhodamine-IgG (Boehringer Mannheim, Indianapolis, IN) in PBS with 1% NGS. Gels were washed
for 1 h in PBS following incubation with each antibody.
Stained gels were mounted in glycerol-PBS and viewed
using a Zeiss LSM confocal microscope equipped with
100 x /1.3 Neofluar oil immersion lens.
Staining of Fixed Preparations
Cultures were fixed for 15 min in CSB containing
2% paraformaldehyde, then extracted for 5 min in CSB
containing 1% Triton X-100 and 4% PEG. Fixed preparations were then washed in PBS and stained for 1 h at
room temperature with 0.3 pM rhodamine phalloidin in
PBS. Stained gels were subsequently washed in PBS and
examined by confocal microscopy as above for immunostaining.
Image Processing
All micrographs were recorded digitally as TIFF
format files and printed using a Fujix Pictrography 3000
thermal transfer printer (Crimson Tech, Cambridge,
MA). Brightness and contrast modifications were made
using Adobe Photoshop image processing software (version 3.0, Adobe Systems Inc., Mountain View, CA).
RESULTS
Electroporationof Fibroblasts in Collagen Gels
Up to 80% of fibroblasts cultured in collagen gels
are loaded with dextran (4 kDa) following electroporation in either DFH (serum free culture medium) or ICEB
(intracellular buffer), using field strengths between 500
and 1,000 Vcm-' and a capacitance of 960 p F (Fig. 1).
The time constants (time required for voltage to decay to
37% of its initial value) generated under these conditions
are between 10 and 20 ms. Field strengths greater than
1.0 kVcm-' detach collagen gels from the supporting
ring of filter paper. Capacitance settings between 125
and 500 pF do not support loading within the range of
field strengths tested. Cell viability, assessed by fluorescein diacetate (FDi) conversion, declines with increasing
voltage from 95% (no electroporation) to 61.7 2 7.4%
(mean k s. d.) for DFH (1,000 Vcm-'), and to 30.0
15.6% for ICEB (1,000 Vcm-'). Parameters for optimal
loading are 875 Vcm-'/960 p F for cells in DFH (viability, 71.7 ? 15.4% of total cells counted; loading, 53.3
2 30.6% of total cells counted) and 750 Vcm-'/960pF
for cells in ICEB (viability, 72.3 2 7.8%; loading, 50.0
*
f 8.9%).ICEB supports electro-loading of fibroblasts in
collagen gels with different molecular weight dextrans
ranging from 4-150 kilodaltons, as well as immunoglobulin (Table I). Non-viable (FDi negative) cells rarely
retain low molecular weight dextran (TRITC-dextran, 4
m a ) . The cytoplasmic distribution of loaded macromolecules is confirmed by live confocal microscopy (Fig. 2).
Effects of Electroporation of Fibroblast Migration
Fibroblasts begin to migrate out of corneal stroma
within 6-8 h after placing the tissue inside a collagen
gel. The traction forces exerted by emigrating fibroblasts
cause a radial alignment of nearby collagen fibers which,
in combination with contact inhibition between the cells,
promotes directed migration of cells away from the explant (Fig. 3). Following mitosis, however, one daughter
cell may return towards the explant until contact inhibition reverses its direction. After 18-24 h in culture, 73%
(n = 45) of cells continue to migrate in a directed manner
0.40 (s.d.) p d m i n .
with an average speed of 0.56
During migration, filopodia (small cylindrical extensions
of the cytoplasm [Trinkaus, 19731) continually extend
and retract from a larger and more stable pseudopodal
extension [Trinkaus, 19731, which is maintained in the
direction of cell migration. Pseudopod branching is often
observed, but additional branches wither and collapse
back towards the cell. Additional filopodia are occasionally displayed at the rear during the approach of neighboring cells, but the forwards movement of cells is usually associated with detachment of cellular material from
the rear. This behavior of control cells is consistent with
that reported previously for corneal fibroblasts migrating
through collagen matrices [Bard and Hay, 19751. Collection of confocal images at intervals of 15 min did not
reveal noticeable changes in the motility of cells as compared with cells monitored by conventional light microsCOPY.
Cells electroporated in culture medium (DFH), display similar filopodial activity to control cells, but in
90% of loaded cells (n = 11) the direction of pseudopodal
extension continually changes in a random manner (Fig.
4). These changes in the direction of pseudopod protrusion mostly occur along the long axis of the cell, but
extensions occasionally emerge at the side of the cell
(Fig. 4; 45 min). Cells electroporated in culture medium
therefore fail to migrate more than 10-15 pm in the first
2 h following their return to culture at 37°C. This behavior is observed following loading of cells in culture
medium with either phalloidin or dextran. Extended observations of cell recovery reveal that 62% of cells electroporated in culture medium display evidence of directed migration after 3 h at 37"C, and 84% by 4 h.
The motility of cells following electroporation in
cytoskeleton stabilizing buffer (CSB; as used for immu-
*
Effects of Electroporation
I 00 $T
T
90
T
I
a
*
II 1
b
T
l T
349
T
*
T
/I
30
20
10
r
L
625
750
40
30
20
r
875
0
4
I 500
1000
0
Vcm-l/960 p F
Fig. 1. Viability and loading of corneal fibroblasts cultured in collagen gels following electroporation in (a) culture medium (DMEM/
F12/20 mM Hepes, pH 7.2) or (b) intracellular electroporation buffer
(ICEB). Viability (white bars) is demonstrated by fluorescein diacetate conversion following electroporation in the presence of TRITC-
1
625
1i
875
750
Vcrr1-~/960pF
1000
dextran (4 m a ) . Loading (black bars) is illustrated by uptake of fluorescent dextran (4 m a ) . Bars represent mean (? standard deviation)
percentage of the total number of cells observed (2100). Asterisks
indicate electroporation parameters used in subsequent studies of cell
motility and the cytoskeleton.
TABLE I. Electroloading of Fibroblasts in Collagen Gels With Dextran and IgG
% Cells loaded, mean (SD)
Molecule
FITC-dextran
40 kDa
70 kDa
150 kDa
FITC-IgG
mg/ml
Control
750 Vcm-’
875 Vcm-’
I ,ooOVcm-’
10
20
40
5
0.5 (0.7)
2.5 (3.5)
1.5 (2.1)
0.0 (0.0)
20.5 (10.6)
65.0 (9.9)
59.5 (3.5)
-
65.0 (1.0)
62.3 (15.0)
67.7 (30.6)
-
52.7 (7.8)
54.3 (15.9)
64.0 (21.2)
41.3 (4.2)
nostaining) was not assessed due to poor viability, but
cells loaded in CSB containing low calcium (1 pM),
instead of EGTA (5 mM), display random pseudopod
activity and, hence, fail to migrate effectively in any
given direction (n = 6). However, following electroporation in intracellular buffer (ICEB), 83% of loaded cells
display directed migration (0.46 0.19 pm/min, n =
18) that is similar to that performed by non-electroporated cells. The opportunity was therefore taken to examine the feasibility of studying changes in the F-actin
cortex of migrating cells following loading of phalloidin
(Fig. 5). Cells electroporated in ICEB containing 3 pM
rhodamine phalloidin initially display a pale, even staining of the F-actin cortex (labeled in Fig. 5 as “C”), but
leading cytoplasmic extensions are poorly stained when
compared with fixed preparations (refer to Fig. 8) due to
new actin polymerization in the time (30-45 min) between loading phalloidin and observing the cells. This
phenomenon has been noted previously for cells micro-
*
injected with phalloidin [Wehland et al., 1977, 19801).
During migration, phalloidin-stained F-actin accumulates at the rear of the cell (labeled “X” in Fig. 5) and at
the base of pseudopodia (labeled “Y” in Fig. 5 ) . Sites of
accumulation at the base of additional pseudopodia remain following the collapse of these extensions, and sites
which develop at the rear remain fixed to the substrate as
the cell migrates forwards.
Effects of Electroporation on the Cytoskeleton
Prior to electroporation, fibroblasts cultured in collagen gels display a dense network of microtubules
which extend the length and breadth of the cell. However, 10 min after electroporation in culture medium
(DFH), the majority of cells (275%) display only a few
microtubules radiating from an organizing center (Fig.
6a). This degree of microtubule disruption is similar to
that produced by treating cells for 1 h at 37°C with 2
350
Harkin and Hay
Fig. 2. Effect of electroporation on uptake of fluorescent dextran by
corneal fibroblasts cultured within collagen gels. Control cells (a,b)
were incubated for 15 min at 25°C in “intracellular” electroporation
buffer containing 10 mglml TRITC-dextran (76 m a ) , and washed for
90 min at 25°C in phenol red-free culture medium. These cells have
failed to take up detectable levels of fluorescent dextran (b). Loaded
cells (c,d) were treated as controls but electroporated at 750 Vcm-’/
960 (LFprior to washing. The cytoplasmic uptake of fluorescent dextran by electroporated cells is confirmed by live confocal fluorescence
(d). Cultures were incubated at 37°C for 30 min prior to image acquisition.
pg/ml nocodazole and is only slightly improved by electroporation in the presence of 2 mM EGTA (data not
shown). In contrast, cells electroporated in either cytoskeleton stabilizing buffer (CSB) or intracellular electroporation buffer (ICEB) display little or no evidence of
microtubule disruption 10 min after electroporation (Fig.
6b,c, respectively). However, microtubules are disrupted when cells are electroporated in ICEB containing
2100 pM calcium chloride.
One hour after returning electroporated cells to culture medium, cells electroporated in DFM display a more
extensive microtubule network than that observed 10 min
after electroporation, but few microtubules extend more
than 10 pm from the organizing center (Fig. 6d). Almost
complete recovery of microtubules is achieved within
3-4 h culture at 37°C. Cells electroporated in CSB dis-
play completely disrupted microtubule networks following their return to culture medium (Fig. 6e). In contrast,
cells electroporated in ICEB show only slight disruption
of their microtubule networks which appears to be
mainly restricted to the outermost parts of the cell (Fig.
6f). However, the degree of microtubule disruption is
increased if cells electroporated in ICEB are maintained
in this buffer for more than 5-10 min prior to their return
to culture medium.
A perinuclear collapse of vimentin intermediate filaments is associated with electroporation conditions
which cause the disruption of microtubules (Fig. 7). This
effect is reversible and follows a similar recovery period
to microtubules. However, F-actin filaments are stable
following electroporation of fibroblasts in culture medium (Fig. 8).
Effects of Electroporation
Fig. 3. Directed migration of chick embryo corneal fibroblasts away
from explant of corneal stroma. This piece of corneal stxoma (bottom
left) from 6-day-old chick embryo was cultured for 24 h inside a
hydrated collagen matrix (1 mg/ml) before fixation with 2% paraformaldehyde in PBS. The orientation of cells, especially those furthest
from the corneal stroma, is indicative of their directed migration away
from the explant prior to fixation.
DISCUSSION
In summary, we demonstrate that corneal fibroblasts cultured within hydrated collagen matrices can be
loaded with dextran (4-150 m a ) , immunoglobulin, and
rhodamine phalloidin, using electroporation parameters
(500-1,000 Vcm-'/960 pF) similar to those previously
reported to permeabilize fibroblasts suspended in medium [Glogauer and McCulloch, 19921. However, the
subsequent motility of these cells within ECM is dependent upon the choice of electroporation buffer, and the
conditions which follow its use. Fibroblasts electroporated in serum-free culture medium fail to perform directed migration, seemingly because pseudopodia are
projected randomly. Microtubules are disrupted and vimentin intermediate filaments adopt a pennuclear distribution, but actin filaments remain intact following electroporation of cells in culture medium. However, cells
electroporated in a buffer which mimics the ionic composition of intracellular fluid (ICEB, 5 10 kM), continue
to migrate through collagen with coordinated pseudopod
formation and display little or no change in their cytoskeleton, when returned to culture medium within 10
min. Thus, ICEB supports loading of cells within collagen gels with low concentrations of rhodamine phalloi-
351
din and facilitates studies of the F-actin cortex during cell
migration through ECM .
We initiated this research, because we believe that
it is important to study the migration of fibroblasts within
a 3-D substrate of ECM. Planar substrata facilitate studies of cell migration by assisting optical resolution of
cellular structures, but the existence of an upper free
surface limits our understanding of how mesenchymal
cells actually migrate when surrounded by ECM in vivo.
Fortunately, developments in confocal microscopy now
provide opportunities for a detailed analysis of the motile
behavior of fibroblasts within more natural 3-D substrata. Indeed, we were able to record the migration of
living fibroblasts in collagen gels at 15-min intervals for
up to 2 h using attenuated laser light and a temperaturecontrolled incubation chamber. However, collagen gels
hamper the microinjection of probes for labeling the cytoskeleton, because the fine injection needles required
often clog or break upon entering the gel. Therefore, in
the present study, we explored the possibility of loading
corneal fibroblasts suspended within collagen gels by
using electroporation.
Electroporation proved to be the method of choice
for introducing macromolecules into the cytoplasm of
cells embedded in ECM. In addition to providing a noninvasive method for loading cells surrounded by ECM,
electroporation loads large numbers of cells in a fraction
of the time required for microinjection of a single cell.
Since electroporation can affect cell viability [Baum et
al., 1994; Wolf et al., 19941, we began our study by
establishing optimal parameters for loading fibroblasts in
collagen gels by this method. We soon found that although chicken fibroblasts are readily loaded in DMEMI
F12 culture medium (DFH), the lack of directed migration for up to 3-4 h following electroporation makes this
medium an unsuitable choice of electroporation buffer
for studies requiring immediate observations of cell migration. For example, cells loaded with rhodamine phalloidin in culture medium may completely redistribute
this F-actin probe before they resume normal migration.
Vasiliev and others [Vasiliev et al., 1970; Bershadsky et al., 1991; Liao et al., 19951 have concluded that
microtubules are necessary for the polarized morphology
and directed migration of fibroblasts attached to planar
substrata. Moreover, it is known that high levels of calcium ions such as those present in the DFH culture medium (-1 mM) inhibit the stability of microtubules in
vitro [Borisy et al., 19761. We therefore considered that
the lack of directed movement displayed by cells in collagen after electroporation in DFH might be due to depolymerization of microtubules following the entry of
Ca2+ ions from the surrounding medium. The correlation that we found between microtubule stabilitylreformation and directed movement clearly supports this idea.
352
Harkin and Hay
Fig. 4. Typical behavior of corneal fibroblast in collagen gel following electropration in culture medium. This cell was electroprated in
DhtEM/F12/20mM Hepes (pH 7.2) containing 3 pM rhodamine phalloidin, then cultured at 37°C for 90 min prior to examination by
confocal microscopy. While pseudopodia (p) and filopodia (f) are
produced, the shifting location of these extensions results in little net
migration. This poor migratory behavior is apparent for 2-3 h following electroporation in culture medium and is also displayed by cells
loaded with fluorescent dextran. Loading of this cell was confirmed by
confocal fluorescence (not shown).
This conclusion is further supported by the fact that cells
display apparently normal directed migration following
electroporation in ICEB which contains low concentrations of Ca2+ ions (1 pM). However, factors other than
calcium may also contribute to the disruption of microtubules since stability was only partially improved in the
presence of EGTA (2 mM). Furthermore, a role for vimentin intermediate filaments in directed migration cannot be ruled out since they collapse to a perinuclear location in the absence of microtubules. It also remains
possible that electroporation may have deleterious effects
on organelles required for directed movement such as the
Golgi apparatus [Bershadsky and Futerman, 19941. Interestingly, collagen fiber lattices do not support directed
migration of fibroblasts in the absence of microtubules as
do grooves cut into silicon wafers [Oakley and Brunette,
19951.
In contrast to microtubules and vimentin filaments,
F-actin filaments are apparently unaffected by electroporation in culture medium (Fig. 8). This finding is not
surprising since cells continue to produce pseudopodia
following electroporation in this medium. Corneal fibroblasts suspended in collagen gels display a dense cortical
(ectoplasmic) network of F-actin filaments surrounding
an endoplasm containing myosin 11, microtubules, and
vimentin filaments [Tomasek et al., 1982; Tomasek and
Hay, 19841. We have suggested that cortical F-actin behind the leading pseudopodium becomes fixed to the
surrounding ECM via integrins, thus allowing the myosin-rich endoplasm supported by microtubules and/or vimentin filaments to slide forwards on the cortex [“fixed
cortex” theory; Hay, 1985, 1989; Bilozur and Hay,
19891. The deposition of cortical cytoplasm containing
plasma membrane, F-actin, talin [Daniels and Hay,
19901, and pl integrins [Regen and Horwitz, 1992; Palecek et al., 19961 in the wake of migrating cells is
Effects of Electroporation
Fig. 5 . Directed migration of corneal fibroblast through collagen following electroporation in intracellular buffer (ICEB). This cell was
electroporated in ICEB containing 1 pM added CaCl, and 3 pM
rhodamine phalloidin, then cultured at 37°C for 30 min prior to examination by confocal microscopy. Note that maintenance of an antenor pseudopod facilitates directed movement of the cell. The actin
cortex (C) is initially stained evenly towards the middle and rear of the
353
cell, but the anterior extensions are poorly stained due to new actin
polymerization. During subsequent forwards migration, phalloidinlabeled actin filaments accumulate at the rear of the cell (X) and near
the base of pseudopodia (e.g., Y). These sites of F-actin accumulation
appear to remain relatively fixed with respect to the surrounding collagen gel.
354
Harkin and Hay
Fig. 6. Effect of electroporation buffer on microtubule stability. Corneal fibroblasts cultured in collagen gels were electroporated in either
serum-free culture medium (DFH), cytoskeleton stabilizing buffer
(CSB; as used for immunostaining), or intracellular buffer (ICEB),
then placed at 37°C for 10 min to assist resealing of plasma mem-
branes. Immunostaining for tubulin was then carried out immediately
(a, DFH; b, CSB; c, ICEB) or 1 h after returning cells to 25°C culture
medium (d,e,f, respectively). Each image is a projected composite of
10 1 pm optical sections. The apparent fragmentation of microtubules
observed in b and c is an artifact of the optical sectioning.
Effects of Electroporation
355
Fig. 7. Electroporation conditions which disrupt microtubules also
cause a perinuclear collapse of vimentin intermediate filaments;
(a) corneal fibroblasts immunostained for vimentin after 48 h culture
inside a hydrated collagen gel; (b) cells immunostained for vimentin
1 h following electroporation in serum-free culture medium. Staining
for F-actin (Fig. 8) reveals that these cells retain bipolar morphology
after electroporation in culture medium.
Fig. 8. Actin filaments are resistent to electroporation in culture medium. This cell was fixed and stained 10 min after electroporation in
serum free culture medium. The phalloidin staining for F-actin in
cytoplasmic extensions suggests that the stability of actin filaments is
unaffected by electroporation conditions which cause disruption of
microtubules and collapse of vimentin networks.
consistent with this theory, as is the fact that actin
mRNA [Lawrence and Singer, 19861 and newly synthesized plasma membrane locate near the leading edge
[Kupfer et al., 19871. Along these lines, it is tempting to
speculate that the lack of directed migration observed in
the absence of microtubules might be mediated by an
inability of the myosin-rich endoplasm to move on the
actin cortex in their absence.
Given the apparent requirement for microtubules
and/or vimentin filaments for directed migration of fibroblasts through collagen lattices, we applied ourselves
to the task of developing the best electroporation proto-
356
Harkin and Hay
col to load cells without adversely affecting the cytoskeleton. Since Pipes/EGTA buffers support the cytoskeleton
when cells are permeabilized with detergent prior to immunostaining, we decided to develop an electroporation
buffer based on these ingredients. The stability of microtubules immediately following electroporation in
CSB initially suggested that this might be a suitable
buffer for our experiments, but unfortunately cells
loaded in this medium displayed disrupted microtubules
when returned to culture medium. The most likely explanation for this result is that CSB does not enable electroporated cell membranes to reseal effectively before
their return to culture medium (DFH). Therefore, while
CSB proved to be an unsuitable electroporation buffer
for studies of directed cell migration, the stability of
microtubules immediately following electroporation in
this buffer at least indicated that microtubules are not
directly disrupted by the electric currents used.
Following studies with CSB, we considered making
several modifications to this buffer including substitution
of EGTA with low calcium (1 pM) and increasing pH. In
the end, we designed a buffer (ICEB) which mimics as
closely as possible the ionic composition of intracellular
fluid (i.e., high potassium, low sodium, and low calcium). The problem of how to add high quantities of
potassium ions without accompanying anions was solved
by neutralizing the required amount of potassium in the
form of KOH with Pipes free acid. While a pH of 6.8 is
optimal for microtubule assembly [Borisy et al., 19761,
preliminary results suggested that a pH of 7.0 improved
the ability of cell membranes to reseal following electroporation. Myo-inositol was used [Neil and Zimmermann,
19931 to adjust osmolarity without changing the ionic
strength or pH. ATP was omitted from the electroporation
medium because it is known to permeabilize plasma membranes [McNeil, 19891 to low molecular weight molecules ( I1 kDa) and may therefore increase the risk of
exposing microtubules to extracellularCa2 ions following the return of cells to culture medium. It should be
noted that while only 1-10 pM of CaCl, were added to
ICEB, these concentrations are less than, or equal to, that
for Ca2+ ions already present in standard preparations of
distilled deionized water. In the end, ICEB is a buffer that
assists electroporation of cell membranes, supports stability of the cytoskeleton, and enables fibroblasts to migrate normally when returned to culture medium.
While other types of intracellular buffers have been
used previously for cell electroporation [Knight and
Scrutton, 19861, we believe that ICEB is the first buffer
to be designed with attention to the requirements for
studies of fibroblast motility. We believe that the confocal and electroporation methods developed in the present
study should promote future studies on the behavior of
the F-actin cortex during the directed migration of fibro+
blasts within ECM. Furthermore, the large size range of
macromolecules loaded in the present study, suggests
that the technique may be suitable for loading cells in
ECM with a variety of other molecules of current interest
including small GTP-binding proteins [Hall, 19941, and
cell lineage markers [Artinger et al., 19951.
ACKNOWLEDGMENTS
We thank Eric Berthiaume, Dr. Joel Swanson, and
Dr. Stephen Baer for their advice on microscopy, Megan
Maultsby for technical assistance, and Dr. Charles
Vanderburg for his helpful comments throughout the
preparation of this article. The E7 hybridoma supernatant
developed by Dr. M. Klymkowsky of the University of
Colorado, and the H5 hybridoma supernatant developed
by Dr. J. Sanes of Washington University School of
Medicine were both obtained from the Developmental
Studies Hybridoma Bank maintained by the Department
of Pharmacology and Molecular Sciences, The Johns
Hopkins University School of Medicine, Baltimore, MD
21205, and the Department of Biology, University of
Iowa, Iowa City, IA 52242, under contract NOl-HD-23144 from the NICHD. This work was supported by
U.S. Public Health Service grants R01-EY09721 and
ROl-DE11142.
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