Journal of Cell Science 105, 167-177 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
167
Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of
plasma membrane vesicles containing actin, annexins II and VI, and b1
integrin receptors
Tien-Ling Lee, Ying-Chun Lin, Katsumi Mochitate* and Frederick Grinnell†
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical School, Dallas, Texas 75235, USA
*Present address: National Institute for Environmental Studies, Tsukuba, Japan
for correspondence
†Author
SUMMARY
To learn about the effects of tension on fibroblast function, we have been studying initial cellular responses to
stress-relaxation. Human foreskin fibroblasts were cultured in anchored collagen matrices for 2 days, during
which time mechanical stress developed. Subsequently,
the matrices were dislodged; thereby allowing stress to
dissipate. Within 5 min after initiating stress-relaxation,
fibroblasts retracted their pseudopodia. At this time, we
observed the disappearance of cellular stress fibers and
the formation of actin clusters along the cell margins.
The actin was found to be located inside 200 nm diameter vesicles that were budding from the cell surface.
Vesicles isolated from the matrix after stress-relaxation
contained prominent 24 kDa, 36 kDa (doublet), 45 kDa,
and 135 kDa polypeptides. The 45 kDa polypeptide was
the major component in the Triton-insoluble vesicle
fraction and appeared to be actin. The 36 kDa (doublet)
polypeptide, which was found predominantly in the
Triton-soluble vesicle fraction, was identified as annexin
II. Vesicles also contained annexin VI and b1 integrin
receptors but not tubulin, vimentin, vinculin or annexin
I. The results suggest that stress-relaxation of fibroblasts induces a novel ectocytotic process involving transient budding of intact, plasma membrane vesicles from
the cell cortex. On the basis of their morphological and
biochemical features, these vesicles may be analogous to
the ‘matrix vesicles’ released by chondrocytes and could
play a role in extracellular matrix remodeling after
wound contraction.
INTRODUCTION
collagen matrix in which they are embedded (Bell et al.,
1979). If the matrix is mechanically anchored during contraction, then mechanical stress develops in response to tension generated by the cells. Actin stress fibers in the cell
cytoplasm become oriented along the long axis of the cells
(Farsi and Aubin, 1984; Unemori and Werb, 1986), and
cells and collagen fibrils become aligned in the same plane
(Nakagawa et al., 1989a). Strain gauge measurements show
that the force generated by fibroblasts under these conditions is comparable to that generated in contracting skin
wounds or during tooth eruption (Kasugai et al., 1990;
Delvoye et al., 1991; Kolodney and Wysolmerski, 1992).
Several studies have shown that the proliferative capacity of fibroblasts in collagen matrices depends in part on
mechanical stress. Cells in contracted collagen matrices that
are under mechanical stress divide in response to growth
factors, but DNA synthesis stops once stress dissipates
(Nakagawa et al., 1989a,b; Fukamizu and Grinnell, 1990).
When fibroblasts contract floating collagen matrices,
mechanical stress does not develop. Under these conditions
the cells show low levels of DNA synthesis (Sarber et al.,
The importance of mechanical force for cell function pertains to a wide range of biological organisms including bacteria and plants. Recent interest in this subject is indicated
by reviews on ‘tensegrity’ (Ingber and Folkman, 1989),
‘mechanogenetic’ (Erdos et al., 1991) and ‘mechanogenic’
(Vandenburgh, 1992) models of cell growth regulation.
Even the well-known dependence of cell growth on cell
shape (Folkman and Moscona, 1978) may turn out to be an
effect of cell tension (Curtis and Seehar, 1978; O’Neill et
al., 1990).
We have been studying the effects of mechanical force
on fibroblasts in three-dimensional collagen matrix cultures.
In these matrices, fibroblasts bind to individual collagen fibrils (Bellows et al., 1982; Allen and Schor, 1983) and surround clusters of fibrils with cell surface extensions (Grinnell and Lamke, 1984). Binding interactions are mediated,
at least in part, by a2b1 integrin receptors (Schiro et al.,
1991; Klein et al., 1991).
During culture, fibroblasts reorganize and contract the
Key words: collagen matrix, ectocytosis, actin, annexin, stress
168
T.-L. Lee and others
1981; Van Bockxmeer et al., 1984; Yoshizato et al., 1985)
and become arrested in G0G1 (Kono et al., 1990). On the
other hand, when external mechanical stress is applied to
collagen matrices containing fibroblasts, DNA synthesis
increases (Jain et al., 1990).
One approach to understanding how mechanical stress
regulates fibroblast function is to characterize the initial
events that occur when fibroblasts under stress are allowed
to relax. Our laboratory and Tomasek’s laboratory independently introduced a culture model designed to permit
this characterization (Mochitate et al., 1991; Tomasek et
al., 1992). In this model, fibroblasts in collagen matrices
anchored on culture dishes were cultured for 2-5 days,
during which time mechanical stress developed. Subsequently, the contracted matrices were dislodged mechanically, thereby initiating stress-relaxation. We report here on
a novel secretory process triggered by stress-relaxation.
Small (approx. 200 nm diameter), right-side out, plasma
membrane vesicles, which contained actin, annexins II and
VI, and b1 integrin receptors, were released from the cells
into the collagen matrix. Details of these findings are
reported here.
blue exclusion. Moreover, fibroblasts harvested from either matrix
were able to attach and spread on culture dishes within several
hours and subsequently proliferated at a similar rate (doubling
time approx. 24 h).
Alternatively, after dispersing cells from the matrices, enzymatic activity could be blocked by the addition of proteinase
inhibitors leupeptin and pepstatin A (1 mg/ml each), and AEBSF
(aminoethylbenzenesulfonyl fluoride, 1 mM; Calbiochem) in
DPBS. This resulted in less contaminating serum proteins being
present in the vesicle preparations.
After centrifugation, proteinase inhibitors (see above) were also
added to the cell-free supernatants. Samples were then centrifuged
for 1 h at 4°C at 100,000 g (Beckman L8-M ultracentrifuge), and
the high-speed pellets (vesicle fraction) were resuspended in
DPBS.
To prepare Triton X-100-soluble and -insoluble fractions of
cells and vesicles, samples were mixed with equal volumes of
extraction buffer containing 2% Triton X-100, 160 mM KCl, 40
mM imidazole-HCl, 1 mg/ml leupeptin and pepstatin A, 1 mM
AEBSF, 20 mM EGTA, and 8 mM sodium azide, pH 7.0, and
incubated for 10 min at 4°C (White et al., 1983). The Triton-insoluble fraction was separated from the soluble fraction by centrifugation for 10 min at 4°C at 12,000 g in an Eppendorf microfuge.
Transmission electron microscopy
MATERIALS AND METHODS
Hydrated collagen matrix cultures
Maintenance of human foreskin fibroblast monolayer cultures and
preparation of hydrated collagen matrices from Vitrogen ‘100’
collagen (Celtrix Labs, Palo Alto, CA) have been described previously (Nakagawa et al., 1989a). Briefly, fibroblasts were added
to the neutralized collagen solutions (1.5 mg/ml) at a concentration of 5 ×105 cells/ml. Samples (0.2 ml) of the cell/collagen mixtures were prewarmed to 37°C for 3-4 min and then placed in
Costar 24-well culture plates. Each sample occupied an area outlined by a 12 mm diameter circular score within a well. Gelation
required 60 min at 37°C, after which 1.0 ml of Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10%
fetal bovine serum (FBS, Intergen Co, Purchase, NY) and 50
mg/ml ascorbic acid was added to each well. It was important not
to add serum until after gel polymerization, otherwise the matrices spontaneously dislodged from the underlying culture surface
during subsequent culture.
Collagen matrices containing fibroblasts were incubated in a
humidified CO2 incubator for 2 days, during which time the fibroblasts contracted the matrix and mechanical stress developed. To
initiate stress-relaxation, matrices were gently dislodged from the
underlying tissue culture substratum with a spatula.
Harvesting cells and vesicles from collagen
matrices
Contracted matrices (anchored or dislodged) were rinsed twice for
10 min at 22°C with DPBS (see below) minus divalent cations
and then incubated for 10 min at 37°C with 0.2 ml of 0.05%
trypsin/0.53 mM EDTA solution (GIBCO) followed by 20-30 min
with 0.25 ml of collagenase solution (5.0 mg/ml Sigma type I collagenase in 130 mM NaCl, 10 mM calcium acetate, 20 mM Hepes,
pH 7.2). After cells dispersed completely, enzymatic activity was
blocked by the addition of 0.05 ml FBS. Fibroblasts were collected by centrifugation at 200 g for 10 min at 22°C (Sorvall GLC1 centrifuge) and resuspended in DPBS (150 mM NaCl, 3 mM
KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1 mM KH2PO4, 6 mM
Na2HPO4, pH 7.2). Greater than 95% of the cells recovered from
anchored or dislodged matrices were viable, as shown by trypan
Collagen matrices to be studied by transmission or scanning electron microscopy were prepared on solvent-resistent Thermanox
plastic coverslips (Nunc Inc., Naperville, IL). Samples for transmission electron microscopy were fixed for 1 h at 22°C with 2%
glutaraldehyde, 4% paraformaldehyde and 1% tannic acid in 0.1
M sucrose, 0.1 M sodium cacodylate-HCl buffer, pH 7.4, and then
washed and post-fixed for 1 h at 22°C in 1% osmium tetroxide
with 1.5% potassium ferrocyanide in the same buffer. After thorough washing with distilled water, samples were dehydrated with
30% ethanol for 10 min at 22°C and stained en bloc with 2%
uranyl acetate in 50% ethanol for 1 h. Subsequently, the samples
were dehydrated, impregnated with pure propylene oxide, and
embedded in pure Epon 812/Araldite mixture. Thin sections (80
nm) were cut with a Reichert-Jung Ultracut ultramicrotome, collected on copper grids without supporting film, and stained with
2% aqueous uranyl acetate for 12 min and Reynold’s lead citrate
for 5 min before observation. All observations and photographs
were made with a JEOL 1200EX transmission electron microscope at 80 kV.
Scanning electron microscopy
Samples for SEM were fixed as described for TEM and postfixed for 90 min at room temperature in 1% osmium tetroxide
and then passed through two cycles of saturated aqueous thiocarbohydrazide and 1% osmium tetroxide (15 min each step),
with thorough washing with distilled water in between steps.
Dehydration was accomplished by a series of 5-min ethanol
washes: 50%, 70%, 90%, 95% and 100%, followed by two 15min changes of absolute ethanol. Dehydrated samples were
immersed in hexamethyldisilazane (HMDS) for 5 min and airdried directly from HMDS. Excess HMDS was blotted dry by
gently touching the samples with a filter paper. To expose the
underside of collagen matrices, samples were dislodged from the
coverslips using a spatula. To expose the interior of the collagen matrices, portions of collagen were peeled away from
anchored matrices using double adhesive Scotch tape. Air-dried
samples were mounted on aluminum SEM stubs with colloidal
graphite and observed without additional metal coating. Specimens were photographed with a JEOL 840 scanning electron
microscope at 20 kV.
Stress-relaxation of fibroblasts
Immunofluorescence microscopy
Collagen matrices to be studied by indirect immunofluorescence
microscopy were prepared on LabTek 2-well chamber slides
(Nunc). Samples were fixed for 15 min at 22°C with 3%
paraformaldehyde in DPBS. Fixed samples were washed twice for
15 min at 22°C with DPBS containing 1% glycine, 1% crystalline
bovine serum albumin (BSA) (Sigma) and then permeabilized
with 2% Nonidet P-40 in DPBS containing 1% BSA for 10 min
at 22°C. To observe the distribution of actin, samples were stained
for 2 h at 37°C with either FITC-phalloidin (Molecular Probes
Inc., Eugene, OR) or mouse anti-actin monoclonal antibody
(Amersham, Arlington Heights, IL), followed by 1 h at 37°C with
FITC-conjugated goat anti-mouse IgM (Cappel, Durham, NC). To
observe the distribution of tubulin or vimentin, samples were
stained for 2 h at 37°C with mouse anti-b-tubulin monoclonal
antibody (a gift from Dr George Bloom, UT Southwestern Medical School) or mouse anti-vimentin monoclonal antibody (Amersham) followed by 1 h at 37°C with FITC-conjugated goat antimouse IgG (Cappel). In some experiments, samples were
counter-stained for 30 min at 37°C with TRITC-conjugated wheat
germ agglutinin (TRITC-WGA, Molecular Probes Inc.), which
permitted identification of cell contours. At the end of the incubations, preparations were washed in DPBS, mounted with
Mowiol, and observed and photographed with a Zeiss Photomicroscope III.
SDS-PAGE and immunoblotting
Samples of cells and vesicles were dissolved in an SDS-containing buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% mercaptoethanol, pH 6.8), and subjected to
SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) on a 4%
stacking/10% resolving gel using a mini-gel apparatus (BioRad,
Richmond, CA). To identify polypeptides by immunoblotting
(Towbin et al., 1979), SDS-gels were washed for 30 min at 22°C
with 25 mM Tris-HCl, 192 mM glycine, 20% methanol, and
polypeptides were transferred onto nitrocellulose filters with a
BioRad mini-transfer apparatus using 100 volts for 1 h at 4°C.
Nitrocellulose filter strips with immobilized proteins were incubated with blocking solution (150 mM NaCl, 20 mM Tris containing 0.2% Tween-20 and 1% bovine serum albumin, fraction
V) for 1 h at 22°C. The strips were then incubated 2 h at 22°C
with mouse monoclonal antibodies against actin, b-tubulin,
vimentin (Amersham) or vinculin (Sigma), rat monoclonal antib1 integrin subunit (mAb 13, a generous gift from Dr Ken
Yamada, NIH), or mouse monoclonal antibodies against annexins
I, II or VI (Zymed Labs, San Francisco, CA). Subsequently, the
samples were incubated with alkaline phosphatase-conjugated
goat anti-mouse IgM or IgG (BioRad) or goat anti-rat IgG
(BioRad) for 1 h at 22°C, and then developed with nitro blue tetrazolium according to BioRad specifications.
Analysis of cells and vesicles by metabolic
radiolabeling
Fibroblasts were metabolically radiolabeled for 2 h with 20-25
mCi/ml [35S]methionine (ICN, specific activity approx. 1300
Ci/mmole) in methionine-free DMEM (GIBCo) supplemented
with 2% FBS and 50 mg/ml of ascorbic acid, and then further
incubated for 2 h with non-radioactive medium (2 medium
changes). Cells and vesicles were prepared from collagen matrices and extracted with Triton X-100 as described above. The samples were dissolved in sample buffer and subjected to SDS-PAGE
using a 10% resolving gel and a 4% stacking gel. After electrophoresis, gels were fixed with 10% TCA, washed with deionized water, and impregnated for 30 min at 22°C with 1 M sodium
salicylate containing 2% glycerol. Gel films were dried using a
BioRad 583 gel drier and exposed to Kodak XAR-2 film.
169
RESULTS
Collagen matrix reorganization and changes in the
cell cytoskeleton initiated by stress-relaxation
Fig. 1 shows inside (A,B) and underside views (C,D) of
collagen matrices immediately before (A,C) and 5 min after
(B,D) the anchored matrices were dislodged from the plastic substratum. During the initial 48 h contraction period,
fibroblasts had developed an extended, bipolar morphology
(A,C). Within 5 min after dislodging the contracted matrices from the substratum (B,D), the uniform appearance of
the matrix was lost. Large furrows developed as a result of
collagen fibril compression when the interconnected network of separate collagen fibrils became more densely
packed. Fibroblasts (arrowheads) switched from a stretched
to a wavy appearance and decreased in length.
The timing of these changes and their uniform appearance, both inside and at the surface of the matrix, suggested
that mechanical release of tension resulted in a synchronous response of the fibroblast population within the matrix.
No longer restrained, the cells retracted their pseudopodia,
thereby causing rapid reorganization of collagen fibrils to
which the pseudopodia were attached. In this case, matrix
contraction appeared to result from a cell-shortening
process clearly different from the previously described reorganization and contraction of anchored collagen matrices
that occurs as fibroblasts extend their pseudopodia (Stopak
and Harris, 1982; Grinnell and Lamke, 1984; Nishiyama et
al., 1988).
Before stress-relaxation, fibroblasts in contracted collagen matrices contained prominent actin stress fibers parallel to the long axis of the cells (Fig. 2A) as well as microtubule and intermediate filament networks (Fig. 2C and E).
Previously we reported that actin stress fibers collapsed
within 1 h after stress-relaxation (Mochitate et al., 1991).
Fig. 2B shows that their disappearance required no more
than 5 min, and at this time we observed actin clusters along
the cell margins and in the matrix near the cells (Fig. 2B).
Comparable clusters of tubulin or vimentin were not seen
(Fig. 2D and F). Thirty minutes later, the clusters could still
be seen in the matrix but were no longer visible at the cell
surface (not shown), suggesting that in response to stressrelaxation, the cells transiently released a portion of their
actin.
Ectocytosis of actin-containing vesicles
Further characterization of actin clusters in fibroblasts
observed after stress-relaxation was accomplished by studying immunostaining of permeabilized and non-permeabilized samples with anti-actin antibodies (Fig. 3A,C). The
cells were counter-stained with TRITC-WGA to reveal cell
contours (Fig. 3B,D). Unless the cells were permeabilized,
little actin immunostaining was observed (A vs C), indicating that the cells were intact and that the actin clusters
were membrane-enclosed. In addition, by comparing actin
and WGA staining, it was evident that actin in the matrix
near the cells occurred in vesicle-like structures (A,B,
arrows). Most vesicles were intact since, without permeabilization, vesicle-associated actin could not be detected
(C,D, boxed area), and the vesicles were right-side-out,
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T.-L. Lee and others
Fig. 1. Changes in collagen matrix organization after initiation of stress-relaxation. Scanning electron micrographs (SEM) of interior
(A,B) and lower surfaces (C,D) of contracted collagen matrices. During contraction, fibroblasts in anchored matrices developed stretched,
bipolar morphology (A,C). Five minutes after being dislodged, the matrices showed compression and folding (B, D) and fibroblasts
(arrowheads) decreased in length by partial retraction of their pseudopodia. Bar, 100 mm.
since they could be stained with TRITC-WGA with or
without permeabilization.
Release of actin-containing vesicles from the cell surface
would not be expected to occur by routine exocytosis, but
might occur by ectocytosis, i.e. triggered release of rightside-out membrane vesicles (Stein and Luzio, 1991). Ultrastructural observations confirmed this possibility. Fig. 4A
shows the typical appearance of mechanically stressed
fibroblasts in contracted matrices: elongated morphology,
relatively smooth surface, and collagen fibrils (col) closely
apposed to the plasma membrane. After the matrices were
dislodged, the cell surfaces became more convoluted (Fig.
4B), and small plasma membrane vesicles containing amorphous material appeared to be budding from the plasma
membrane. Some of these approx. 200 nm diameter vesicles were still attached to the cells by thin stalks (Fig. 4B,
arrows), and some vesicles appeared to be associated with
collagen fibrils.
In studies on fibroblast motility, Chen (1981) published
electron micrographs showing the formation of similar
vesicular structures at the cell surface during retraction of
the cell’s trailing edge. Perhaps, because the cells were in
monolayer culture, release of the vesicles went unnoticed.
Analysis of vesicles harvested from collagen
matrices
The above studies indicated that stress-relaxation triggered
ectocytosis of plasma membrane vesicles. Since the fibroblasts were embedded in an extracellular matrix, the vesicles
had the potential to accumulate. This possibility was confirmed, and vesicles were recovered from the collagen
matrix by a three-step procedure: enzymatic digestion using
a combination of trypsin and collagenase, low-speed centrifugation to remove cells, and high-speed centrifugation
to separate vesicles from soluble components. Fig. 5 shows
that the high-speed pellet obtained by this procedure contained a mixture of membrane vesicles and fragments. The
vesicles were similar in size and content to those seen bud-
Stress-relaxation of fibroblasts
171
Fig. 2. Changes in cytoskeletal organization after initiation of stress-relaxation. Immunofluorescence micrographs showing the
distribution of actin detected by FITC-phalloidin (A,B), tubulin (C,D) and vimentin (E,F) in whole mounts of collagen matrices
immediately before and 5 min after stress-relaxation. Fibroblasts in anchored matrices contained prominent stress fibers oriented parallel
to the long axis of the cells as well as prominent microtubule and intermediate filament networks. After the matrices were dislodged,
stress fibers depolymerized, and actin clusters appeared along the cell periphery and in the collagen matrix. Bar, 20 mm.
ding from the plasma membrane. Membrane fragments, on
the other hand, may have been derived from collapsed
vesicles.
Cytoskeletal components and b1 integrins in cell and
vesicle fractions were studied by immunoblotting with
monoclonal antibodies against actin, tubulin, vimentin, vin-
172
T.-L. Lee and others
Fig. 3. Appearance of actin-containing vesicles after initiation of stress-relaxation. Immunofluorescence micrographs showing the
appearance of actin-containing vesicles in dislodged collagen matrices. Permeabilized (A,B) or non-permeabilized (C,D) whole-mounts
of collagen matrices 5 min after stress-relaxation were stained with mouse anti-actin monoclonal antibody followed by FITC-conjugated
goat anti-mouse IgM and counter-stained with TRITC-WGA as described in Materials and Methods. Contours of cells and vesicles were
delineated by TRITC-WGA staining in both permeabilized (arrows in B) and non-permeabilized (boxed area in D) samples. The
corresponding FITC-positive, actin-containing vesicles could usually be seen in permeabilized samples (arrows in A) but not samples
without permeabilization (boxed area in C). Bar, 20 mm.
culin and b1 integrin subunit. Fig. 6 shows that the
vesicles contained actin and b1 integrins but excluded tubulin, vimentin and vinculin. These results are consistent with
the immunofluorescence findings and show that only a
selected subset of cellular components sorted into the vesicles, and were probably derived from the cell cortex. The
presence of b1 integrin receptors on the vesicles could
account for their association with collagen fibrils (see
above).
Control experiments (Fig. 7) showed that cells contained
tubulin primarily in the Triton-soluble cell fraction and
vimentin mostly in the Triton-insoluble cell fraction. Actin
was found about equally partitioned in Triton-soluble and
-insoluble fractions of cells and vesicles, which indicated
that the vesicles contained a mixture of G- and F-actin. The
identity of two bands detected with antibodies against b1
integrin is unknown but they may result from the enzyme
treatments used during the harvesting procedure.
Immunoblotting of cell extracts prepared from fibroblasts
in collagen gels without enzymatic treatment revealed only
a single b1 integrin subunit. Similarly, fibroblasts harvested
from monolayer cultures showed primarily the 53 kDa
vimentin band (data not shown).
Analysis of vesicles released from radiolabeled
fibroblasts
To learn more about the vesicles, studies were carried out
with radiolabeled fibroblasts. Cells in mechanically stressed
matrices were incubated in medium containing [35S]methionine for 2 h and then chased for 2 h in fresh medium. Subsequently, the matrices were dislodged. Table 1 shows the
results from three separate experiments. At the end of the
2 h chase period, the vesicle fraction averaged 2.0% of the
total radioactivity. Five minutes after stress-relaxation,
Stress-relaxation of fibroblasts
173
Fig. 4. Changes in fibroblast surface appearance after initiation of stress-relaxation. Transmission electron micrograph of fibroblasts in
contracted collagen matrix before stress-relaxation (A), and 5 min after stress-relaxation (B). Before stress-relaxation, the fibroblast had a
relatively smooth plasma membrane and abundant cell organelles. After stress-relaxation, the surface of the fibroblast became irregular.
Numerous membrane-enclosed vesicles were observed emerging from the plasma membrane and connected by thin stalks to the plasma
membrane (arrows). Also, membrane vesicles could be seen in the matrix, often in close association to collagen fibrils. m, mitochondria;
g, Golgi apparatus; er, rough-surfaced endoplasmic reticulum; col, collagen fibrils. Bar, 200 nm.
Table 1. Quantification of radioactivity in cells and
vesicles before and after stress-relaxation
Anchored matrix
Exp.
Fraction
c.p.m.×10−4
1
Cells
Vesicles
265
6
2
Cells
Vesicles
291±68
4±1
3
Cells
Vesicles
85±7
2±1
Dislodged matrix
% Total*
c.p.m.×10−4
% Total*
2.3
221
24
9.8
1.4
219±3
18±8
7.6
2.2
64±2
6±1
8.3
[35S]methionine
After 48 h of contraction, fibroblasts were labeled with
for 2 h. Following a 2 h chase incubation, some of the matrices were
dislodged. Fibroblasts within anchored or dislodged matrices were
harvested and vesicles were isolated. Samples of the cell and vesicle
fractions were analyzed for radioactivity. Data showing means and s.d.
were from triplicate samples.
*Total=cells + vesicles.
Fig. 5. Ultrastructure of isolated vesicles: transmission electron
micrograph of vesicles harvested by enzyme digestion of collagen
matrices followed by low- and high-speed centrifugation. Many
vesicles had a morphology similar to those observed in Fig. 4B;
others appeared to have collapsed during preparation. There was
little or no contamination of the pellets with cell organelles. Bar,
200 nm.
radioactivity found in the vesicle fraction increased to 8.6%
of the total. Therefore, taking into account the different
incubation times (2 h vs 5 min), the rate of release of
radioactivity from the cells increased almost 100-fold after
stress-relaxation.
Fig. 8 shows analyses of the radiolabeled cell and vesicle fractions by SDS-PAGE and autoradiography. The
polypeptide profile of cells from anchored matrices (AC)
was essentially identical to that of cells from dislodged
matrices (DC), indicating that no cellular proteins were
selectively lost as a consequence of stress-relaxation. In
vesicles isolated after stress-relaxation (DV), several
174
Anti-:
T.-L. Lee and others
act.
C
tub.
V
C
vinc.
β1-int.
C
C
vim.
V
C
V
V
V
kDa
200
116
97
66
45
Fig. 6. Cytoskeletal and b1 integrin composition of cells and
vesicles. Lysates of cells harvested from anchored matrices (C)
and vesicles isolated after stress-relaxation (V) were analyzed by
immunoblotting with antibodies against actin, tubulin, vimentin,
vinculin and b1 integrin. Vesicles contained actin and b1 integrin,
but excluded tubulin, vimentin, and vinculin. Cells or vesicle
samples from one collagen matrix are shown in each lane.
Anti-:
act.
C
Triton-soluble
tub.
vim.
V
C
V
C
V
Triton-insoluble
act.
tub.
vim.
C
V
C
V
C
V
kDa
200
116
97
66
45
Fig. 7. Cytoskeletal distribution in Triton-soluble and Tritoninsoluble fractions prepared from cells and vesicles. Lysates of
cells harvested from anchored matrices (C) and vesicles isolated
after stress-relaxation (V) were extracted with Triton X-100,
centrifuged, and samples were immunoblotted with antibodies
against actin, tubulin and vimentin. Actin was detected at similar
levels in the Triton-soluble and -insoluble fractions. Tubulin was
found primarily in the Triton-soluble cell fraction. Vimentin was
found predominantly in the Triton-insoluble cell fraction. Cell or
vesicle samples from one collagen matrix are shown in each lane.
[35S]methionine-labeled polypeptides appeared to be
enriched compared to the cells, in particular those migrating at 24 kDa, 36 kDa (doublet), 45 kDa and 135 kDa. The
24 kDa and 136 kDa components have yet to be identified,
but the 36 kDa polypeptide was tentatively identified as
annexin II (see below). Except for actin, all of these com-
ponents partitioned primarily in the Triton-soluble fraction.
In the Triton-insoluble fraction of the vesicles actin
appeared to be the major component, whereas actin and a
53 kDa polypeptide (vimentin) were the major components
of the Triton-insoluble fraction prepared from cells.
Fig. 8 also shows that the polypeptide profile of vesicles
from anchored matrices (AV) was essentially identical to
that of vesicles from dislodged matrices (DV). This result
suggests that ectocytotic vesicles were released at a slow
rate from fibroblasts in anchored matrices, perhaps as a
byproduct of cell motility, which would be consistent with
the quantitative data (Table 1).
Analysis of annexins in vesicles harvested from
collagen matrices
Several laboratories have reported release of approx. 200
nm diameter plasma membrane vesicles by chondrocytes
(Anderson, 1984). These so-called ‘matrix vesicles’, which
induce biomineralization of cartilage matrix, contain prominent 36 kDa (doublet) and 45 kDa components. The 45 kDa
component has been identified as actin (Muhlrad et al.,
1982; Morris et al., 1992) and the 36 kDa (doublet) component has been identified as a mixture of annexins (Genge
et al., 1992). Annexin VI also has been found in chondrocyte matrix vesicles (Wu et al., 1992). We considered the
possibility, therefore, that the 36 kDa (doublet) polypeptide
in plasma membrane vesicles released from fibroblasts
might correspond to one of the annexins. Fig. 9 shows samples of cells and vesicles isolated from the collagen matrix
and immunoblotted with antibodies against annexins I, II
and VI. All three annexins were detected in the cell lysates,
and annexins II and VI were found in the vesicles.
DISCUSSION
To learn about the effects of tension on fibroblast function,
we have been studying a stress-relaxation model. Within 5
min after initiating stress-relaxation, fibroblasts retracted
their pseudopodia and pulled collagen fibrils together.
Although the contractile mechanism accounting for stressrelaxation is unknown, contraction required the presence of
a serum factor and could be inhibited by cytochalasin D
(Tomasek et al., 1992) or EGTA (Lee and Grinnell, unpublished observation), indicating that it is an active cell-mediated process rather than a consequence of passive elastic
recoil. Stress-relaxation resulted in disappearance rather
than shortening of stress fibers, and therefore did not appear
to result from a myofibril-like contraction (Isenberg et al.,
1976; Kreis and Birchmeier, 1980; Burridge, 1981).
At the same time as the disappearance of stress fibers,
clusters of actin occurred along the cell margins and in the
matrix near the cells. The actin did not appear to be released
from dying cells, but rather was found inside vesicles that
were budding from the cell surface. These regularly shaped,
approx. 200 nm diameter vesicles were membraneenclosed, right-side-out and contained roughly similar proportions of G- and F-actin. After 5 min, some plasma membrane vesicles were connected to the plasma membrane by
thin stalks; others could be found in the collagen matrix,
often bound to collagen fibrils. By 30 min, vesicles were
Stress-relaxation of fibroblasts
Tritonsoluble
Whole
extract
A
C
D
C
A
V
D
V
A
C
D
C
A
V
Tritoninsoluble
D
V
A
C
kDa
200
97
66
45
31
Antiannexin
I
II
VI
DC DV DC DV DC DV
kDa
97
66
45
31
21
14
Fig. 9. Annexins I, II and VI in cells and vesicles. Lysates of cells
harvested from dislodged matrices (C) and vesicles isolated after
stress-relaxation (V) were immunoblotted with antibodies against
annexin I, II and VI. All three annexins were found in the cells, but
the vesicles contained primarily annexins II and VI. The presence
of two annexin II bands in the vesicle fraction may reflect limited
proteolysis during harvesting of the vesicles. Cells or vesicle
samples from one collagen matrix are shown in each lane.
D
C
A
V
D
V
175
Fig. 8. Newly synthesized components in
actin-containing vesicles. SDS-PAGE and
autoradiography of radiolabeled cell and
vesicle proteins synthesized in the
experiment described in Table 1. AC,
anchored cells; DC, dislodged cells; AV,
anchored vesicles; DV, dislodged
vesicles. Several [35S]methionine-labeled
polypeptides appeared to be enriched in
vesicles
compared to the cells, in
* 135
particular components migrating at 24
kDa, 36 kDa (doublet), 45 kDa and 135
kDa. Except for actin, all of these
components partitioned primarily in the
* Vimentin Triton-soluble fraction. In the Tritoninsoluble fraction of vesicles, actin
appeared to be the major component,
whereas actin and a 53 kDa polypeptide
* Actin
(vimentin) were the major components of
* 36
the Triton-insoluble fraction prepared
from cells. Vesicle samples from 0.75
collagen matrix and cell samples from
* 24
0.05 collagen matrix (whole extract and
Triton-soluble fraction) or 0.16 collagen
matrix (Triton-insoluble fraction) are
shown in each lane.
no longer observed on the cell surface but could still be
found in the matrix, suggesting that the budding process
triggered by stress-relaxation was transient.
Taken together, these features indicate that budding triggered by stress-relaxation is an example of what has been
called cellular ‘ectocytosis’ (Stein and Luzio, 1991). Noticing fibroblast ectocytosis would have been difficult had the
cells been in monolayer culture because the vesicles would
have been released into the medium. As already mentioned,
Chen (1981) published electron micrographs showing the
formation of small, vesicular structures on the cell surface
during retraction of the cell’s trailing edge, but release of
vesicles went unnoticed. Having the fibroblasts in a collagen matrix, however, allowed the vesicles to accumulate.
Although the rate of vesicle released increased dramatically
during stress-relaxation, a slow release of vesicles also
appeared to occur in anchored matrices, perhaps as a
byproduct of cell migratory activity.
Analysis of ectocytotic vesicles isolated from the matrix
after stress-relaxation showed that they had a very specific
cytoskeletal composition, i.e. they contained actin but not
tubulin, vimentin or vinculin. These results suggested that
the vesicles were selectively derived from the cell cortex.
The vesicles also contained annexins II and annexin VI, and
annexins normally are localized in the cell cortical region
(Semich et al., 1989; Nakata et al., 1990). Annexin II has
been implicated in the membrane fusion event during exocytosis (Ali et al., 1989; Zaks and Creutz, 1990) and
annexin VI has been implicated in coated vesicle formation
during receptor-mediated endocytosis (Lin et al., 1992), so
the annexins may play a role in the membrane reorganization required for re-sealing ectocytotic vesicles after they
form.
Previous studies indicated that ectocytosis may result
176
T.-L. Lee and others
from disruption of the cell’s cortical cytoskeleton (reviewed
by Gores et al., 1990). One possibility, suggested for agonist-induced ectocytosis of platelet membrane vesicles, is
that activation of the cytoplasmic proteinase calpain results
in degradation of the cortical cytoskeleton components that
stabilize the plasma membrane (Fox et al., 1990), but this
theory has been controversial (Wiedmer et al., 1990). In our
studies we did not observe differences in high molecular
mass components in cells harvested from collagen matrices
before and after stress-relaxation.
Another possibility is that the vesicles represent sites at
which cells were attached to collagen fibrils before stressrelaxation, sites that were released as the cells retracted,
and which then re-sealed to form vesicles. Association of
b1 integrins with the vesicles could account for their adhesion to collagen (Schiro et al., 1991; Klein et al., 1991).
Also, both annexins II and VI have been reported to be collagen-binding proteins (Wirl et al., 1990; Wu et al., 1992).
Regardless of whether the vesicles are released and then
bind to collagen fibrils or are sites of collagen fibril attachment that re-seal after release, the ectocytotic mechanism
provides a means by which cells can export cytoplasmic
proteins that lack a signal sequence. An analogous mechanism has been suggested to occur during the normal course
of myoblast development (Cooper and Barondes, 1990).
In the case of chondrocytes, ectocytosis results in release
of approx. 200 nm ‘matrix vesicles’ that are involved in
matrix remodeling, i.e. these vesicles initiate the biomineralization process (Anderson, 1984). Release of matrix vesicles depends on actin depolymerization (Hale and Wuthier,
1987), and the isolated vesicles have been found to contain
prominent 45 kDa and 36 kDa (doublet) components identified as actin (Muhlrad et al., 1982; Morris et al., 1992)
and a mixture of annexins (Genge et al., 1992). Matrix vesicles also contain annexin VI (Wu et al., 1992).
The physiological function of fibroblast matrix vesicles
is probably unrelated to mineralization because, unlike
chondrocyte matrix vesicles, fibroblast vesicles do not contain detectable alkaline phosphatase (Lee and Grinnell,
unpublished observation). One possibility is that fibroblast
matrix vesicles play a role in tissue remodeling that begins
after the contraction phase of wound repair (Peacock, 1984;
Clark, 1985). Vesicles released by platelets have been
shown to regulate hemostasis by activating the enzyme prothrombinase (Fox et al., 1990; Wiedmer et al., 1990). Vesicles released from fibroblasts may play an analogous role
in activation of metalloproteinases (Mignatti et al., 1988;
He et al., 1989). Interestingly, fibroblasts show activation
of procollagenase in vitro after contraction of collagen
matrices (Mauch et al., 1989; Nakagawa et al., 1989b).
Future studies with isolated vesicles should provide insights
into this possibility.
These studies were supported by NIH grant GM31321. We are
indebted to Dr William Snell for his advice during the course of
this research and to Drs Richard Anderson and Ellis Golub for
their help during preparation of the manuscript.
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(Received 11 November 1992 - Accepted 9 February 1993)