734 | P. D. Arora et al. Molecular Biology of the Cell
MBoC | ARTICLE
Collagen remodeling by phagocytosis is
determined by collagen substrate topology
and calcium-dependent interactions of gelsolin
with nonmuscle myosin IIA in cell adhesions
P. D. Aroraa, Y. Wanga, A. Bresnickb, J. Dawsonc, P. A. Janmeyd, and C. A. McCullocha
aMatrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada; bDepartment
of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; cMolecular and Cellular Biology, University
of Guelph, Guelph, ON N1G 2W1, Canada; dInstitute for Medicine and Engineering, University of Pennsylvania,
Philadelphia, PA 19104
ABSTRACT We examine how collagen substrate topography, free intracellular calcium ion
concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at
collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on pla-
nar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of
gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fi-
broblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and
Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant in-
tracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhe-
sions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to
full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA,
which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to col-
lagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes
enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA
in turn enables actin remodeling and enhances collagen degradation by phagocytosis.
Deregulation of collagen degradation leads to imbalances of matrix
homeostasis (Perez-Tamayo, 1978). These imbalances can manifest
as destruction of normal matrix structure, tissue overgrowth, or
fibrosis in a wide variety of connective tissue lesions that include,
respectively, osteoarthritis, gingival hyperplasia, or heart failure.
Collagen degradation is mediated by an extracellular matrix metal-
loproteinase–dependent extracellular pathway and by a poorly
defined, intracellular phagocytic pathway that involves fibroblasts
(Everts et al., 1996). During phagocytosis, fibroblasts bind collagen
via integrins, which is followed by collagen fibril segregation and
then partial digestion of the fibrils by extracellular matrix metallo-
proteinases (Murphy and Nagase, 2008). Short fibrils are internal-
ized and further digested in lysosomal compartments (ten Cate,
1972; Melcher and Chan, 1981; Everts et al., 1996). It is not under-
stood how cells sequester collagen fibrils and focus their proteolytic
machinery to enable collagen digestion by phagocytosis. Fibro-
blasts in vivo exhibit marked alterations of cell shape around colla-
gen fibrils (ten Cate, 1972; Svoboda et al., 1979; Melcher and Chan,
1981) that might be important for enabling collagen digestion, but
it is not known how cells recruit cytoskeletal proteins to collagen
fibril attachment sites for initiating and enabling phagocytosis.
We considered that the complex topography exhibited by col-
lagen fibrils in vivo might hamper our ability to define the control
systems that regulate collagen phagocytosis. Examination of colla-
gen remodeling by phagocytosis has been primarily studied on cells
plated on rigid, planar substrates. The flattened appearance of
Received: Oct 18, 2012
Revised: Dec 21, 2012
Accepted: Jan 8, 2013
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-10-0754) on January 16, 2013.
Address correspondence to: C. A. McCulloch (firstname.lastname@example.org).
Abbreviations used: [Ca2+]i, free intracellular calcium ion concentration; EGTA,
ethylene glycol tetraacetic acid; GsMTx-4, Grammostola spatulata mechanotoxin
4; GST, glutathione S-transferase; NMMIIA, nonmuscle myosin IIA; Q, quiescent
cells; S, spreading cells; WT, wild type.
© 2013 Arora et al. This article is distributed by The American Society for Cell Bi-
ology under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
Supplemental Material can be found at:
Volume 24 March 15, 2013 Collagen adhesions | 735
It is not understood how NMMIIA regulates adhesion formation to
collagen in more topologically complex environments than planar
cell culture surfaces.
NMMIIA is an actin-based motor protein that is important for cell
migration because of its effect on adhesion, lamellar protrusion, and
polarity (Conti and Adelstein, 2008; Vicente-Manzanares et al.,
2009). Myosin II molecules are hexamers composed of myosin
heavy-chain dimers and two pairs of myosin light chains (Conti and
Adelstein, 2008). The heavy chains include the α-helical coiled-coil
rod domain, which contributes in part to the assembly of nonmuscle
myosin monomers into filaments. The heavy chains also contain
phosphorylation sites for several kinases (Dulyaninova et al., 2005).
In the context of phagocytosis, myosin-superfamily motor proteins
interact with actin filaments to generate contractile forces, which are
required for phagosome development (Groves et al., 2008). It is not
understood how NMMIIA contributes to the regulation of cell adhe-
sion in collagen phagocytosis.
Efficient collagen phagocytosis requires actin assembly at matrix
adhesion sites (Arora et al., 2008a), a process that depends on the
function of the actin-severing and -capping protein gelsolin (Arora
et al., 2003, 2005). Gelsolin is an actin-binding protein that nucle-
ates filament assembly in a Ca2+-dependent manner (Yin and
Stossel, 1979; Yin et al., 1981; Weeds and Maciver, 1993) and plays
an important role in FcγR-mediated phagocytosis but not in comple-
ment-receptor–mediated phagocytosis (Serrander et al., 2000).
Gelsolin associates with NMMIIA at collagen-binding sites; this as-
sociation is required for actin assembly and adhesion to collagen
(Arora et al., 2011), but it is not known whether gelsolin interacts
with NMMIIA directly and how this interaction is regulated to affect
adhesion to collagen.
In this study we examine the effect of collagen substrate topog-
raphy on adhesion formation. We define the role of Ca2+ in regulat-
ing the interaction of NMMIIA with gelsolin to enable adhesion for-
mation and collagen degradation by phagocytosis. With the use of
purified, full-length, and isolated domains of NMMIIA and gelsolin
we show that gelsolin interacts with the coiled-coil domain of
NMMIIA in a Ca2+-dependent manner. This interaction controls actin
filament assembly (Arora et al., 2011) and contributes to adhesion
and phagocytosis of collagen.
Gelsolin and NMMIIA localize selectively to
We determined whether cell adhesion to collagen was affected by
topology. Wild-type fibroblasts plated on collagen-coated planar
surfaces exhibited prominent vinculin staining at focal adhesions.
Colocalization of gelsolin or NMMIIA with vinculin was minimal, as
quantified by the Pearson r (Figure 1A). In contrast, cells plated on
collagen-coated beaded surfaces (2 μm diameter) showed marked
colocalization of gelsolin or NMMIIA with vinculin at bead adhesion
sites (Figure 1B). We estimated the relative abundance of β-actin,
gelsolin, and NMMIIA associated with collagen beads by first count-
ing the number of beads associated with cells, preparing collagen
bead–associated proteins (Glogauer et al., 1998), and then compar-
ing blot densities of the bead-associated proteins with known
amounts of purified gelsolin and NMMIIA that were analyzed on the
same gels. These data indicated that on a molar basis, there was
approximately twofold more gelsolin than NMMIIA that was associ-
ated with the collagen adhesions (Figure 1C; p < 0.05). Gelsolin and
vinculin localized to collagen beads in NMMIIA wild-type embryonic
stem cells but not in NMMIIA-null embryonic stem cells (Figure 1D),
indicating that gelsolin localization to collagen bead adhesions
fibroblasts plated on planar surfaces differs substantially from their
in vivo appearance, which is a more dendritic and stellate shape
than that of cultured cells (Grinnell, 2003). The differences in the
shape of fibroblasts cultured on planar surfaces compared with fi-
broblasts in mammalian connective tissues reflect, in part, the re-
sponse to substrate stiffness (Yeung et al., 2005) and substrate to-
pography (Curtis and Wilkinson, 1998). Indeed, substrate stiffness is
an important determinant of the shape and function of adhesions
that form when cells attach to extracellular matrices (Vicente-
Manzanares and Horwitz, 2011). In the context of substrate topog-
raphy, when avidly phagocytic cells are plated on flat surfaces, they
spread and engage the surface in an attempt to engulf it or until all
of the adhesion receptors are engaged or internalized (Takemura
et al., 1986). A state of “frustrated” phagocytosis can also be
achieved by incubating cells with polystyrene beads of sufficient
size so that the cells are unable to engulf the beads (Cannon and
Swanson, 1992). Accordingly, the topographical presentation of col-
lagen to phagocytic cells might affect the nature of adhesion forma-
tion and possibly collagen phagocytic processes (Grinnell and
Geiger, 1986). We consider here that the topological complexity
of collagen fibril presentation to cells might also affect calcium sig-
naling, which is known to be important for phagocytic processes
(Stendahl et al., 1994; Zimmerli et al., 1996). In particular, previous
data show the existence of stretch-sensitive, cation-permeable
channels in fibroblasts from periodontium (Arora et al., 1994;
Glogauer et al., 1995). Accordingly, the topology of collagen fibril
presentation, which depends on surface characteristics, might affect
the opening probability of stretch-activated, calcium-permeable
channels that regulate intracellular calcium concentration.
Initial cell attachment to collagen in cultured cells occurs via spe-
cialized adhesions and their potential in vivo correlates, fibronexus
junctions (Singer et al., 1984). Focal adhesions are paradigmatic ex-
amples of the complex molecular architectures that enable adhe-
sion receptors like integrins to connect the extracellular matrix to
the actin cytoskeleton (Burridge and Chrzanowska-Wodnicka, 1996).
In cultured cells, adhesions form initially near the leading edge of
the lamellipodium, a region of the cell in which rapid actin polymer-
ization drives membrane protrusion (Pollard and Borisy, 2003). The
earliest adhesions that form after cell contact with matrix ligands are
largely immobile (Alexandrova et al., 2008), but these adhesions can
mature and enlarge over time as the cells become more adherent to
the substrate and more structural and signaling molecules are re-
cruited to the adhesions. In this context, focal adhesions are a het-
erogeneous group of adhesions with a wide spectrum of shapes and
sizes that reflect the temporal continuum of adhesion maturation
(Geiger and Yamada, 2011). They are often small, highly circum-
scribed structures that contain integrins, actin-binding proteins such
as talin and vinculin, and signaling molecules such as FAK and
p130CAS (Geiger et al., 2009).
The maturation of focal complexes to focal adhesions is a pro-
cess that involves the interactions of actin filaments with a large
number of actin-binding proteins such as α-actinin and nonmuscle
myosin IIA (NMMIIA; Choi et al., 2008). Of note, the contractile ac-
tivity of NMMIIA is dispensable for the adhesion maturation process
(Choi et al., 2008) and is also not required for collagen phagocytosis
(Arora et al., 2008a). Although most of our understanding of adhe-
sion formation and maturation has been obtained from the study of
cells cultured on planar surfaces, cells in vivo adhere to and remodel
collagen (Melcher and Chan, 1981) that is organized into complex
surfaces in three dimensions. Indeed, adhesions formed in three-
dimensional matrices tend to be small and highly dynamic and are
NMMIIA dependent (Fraley et al., 2010; Kubow and Horwitz, 2011).
736 | P. D. Arora et al. Molecular Biology of the Cell
Coomassie-stained SDS–polyacrylamide gels of the pelleting as-
says, high-salt (600 mM KCl) buffer prevented NMMIIA binding to
gelsolin that was bound to glutathione S-transferase (GST)–Sephar-
ose beads (Figure 2B). We extended these studies by mapping the
gelsolin-binding domain on NMMIIA filaments using GST pull
downs. GST-gelsolin domains G1–G3 exhibited Ca2+-independent
binding to full-length NMMIIA, whereas gelsolin domains G4–G6
displayed Ca2+-dependent binding to NMMIIA filaments (Figure
2C). Preparations of NMMIIA and gelsolin that were prepared for
immuno–electron microscopy showed increased immunogold label-
ing of gelsolin on NMMIIA filaments compared with preparations
with no primary antibody (Supplemental Figure 1E).
We performed more-detailed binding experiments in which we
used full-length G1–G6, the N-terminal half of gelsolin (G1–G3), the
C-terminal half of gelsolin (G4–G6), and the rod domain of NMMIIA.
Binding experiments were performed in the presence or absence of
EGTA (1 mM). Quantification of the binding of gelsolin to NMMIIA
showed that gelsolin G1–G6 binds tightly to NMMIIA (dissociation
might involve NMMIIA. We immunostained gelsolin-null and wild-
type (WT) cells for the α2 integrin, which is an important integrin
subunit for collagen binding. These results showed colocalization of
α2 integrin with NMMIIA in gelsolin WT cells (but not gelsolin-null
cells) at collagen bead–binding sites (Supplemental Figure 1A).
Interaction of NMMIIA with gelsolin
The finding of colocalization of gelsolin and NMMIIA suggested po-
tential interactions between these proteins. We examined these in-
teractions using purified full-length NMMIIA (Supplemental Figure
1B) and full-length gelsolin. In the preparation of purified NMMIIA,
treatment with MgATP dissociated actin contaminants, as shown by
immunoblotting of the purified fraction for β-actin (Supplemental
Figure 1C). From the purified preparations, negatively stained NM-
MIIA filaments were visualized by transmission electron microscopy
(Supplemental Figure 1D). Pelleting assays showed that full-length
NMMIIA associates with full-length gelsolin in calcium buffer but not
in ethylene glycol tetraacetic acid (EGTA) buffer (Figure 2A). In
FIGURE 1: (A) Representative images of gelsolin and NMMIIA show minimal colocalization with vinculin-stained focal
adhesions in wild-type cells plated on collagen-coated planar substrates. There was 30 and 40% colocalization,
respectively, between gelsolin and vinculin and between NMMIIA and vinculin. (B) Wild-type cells plated on 2-μm
collagen-coated beads show colocalization of gelsolin and NMMIIA with vinculin at collagen bead adhesion sites. Insets
show targeting of gelsolin and NMMIIA to developing phagosomes. (C) Quantitative immunoblot analysis of collagen
bead–associated proteins. Data are mean ± SEM (in picomoles per cell) of β-actin, gelsolin, and NMMIIA. (D) Gelsolin
strongly colocalizes with vinculin at collagen bead–binding sites in wild-type embryonic stem cells but not in NMMIIA-
null embryonic stem cells.
Volume 24 March 15, 2013 Collagen adhesions | 737
FIGURE 2: (A) Pelleting assays show association of full-length NMMIIA with full-length gelsolin in presence or absence
of EGTA. (B) Coomassie-stained SDS-polyacrylamide gel shows that high-salt (600 mM KCl) buffer prevents NMMIIA
binding to gelsolin bound to GST-Sepharose beads. (C) Association between full-length NMMIIA and gelsolin domains
G1–G3 and G4–G6 in pull-down assays. Pellets were visualized on Coomassie-stained SDS–PAGE gel and show that the
interaction between GST-gelsolin domains G4–G6 and full-length NMMIIA requires Ca2+. (D) Quantification of the
binding signal of gelsolin to NMMIIA shows that gelsolin (G1–G6) binds to NMMIIA efficiently with Kd = 0.158 ±
0.082 μM. (E) In the presence of EGTA the binding was reduced (Kd = 0.423 ± 0.152 μM). (F, G) The N- and C-terminal
halves G1–G3 and G4–G6 showed binding with Kd = 0.220 ± 0.049 and 0.212 ± 0.063 μM, respectively. (H, I) In the
presence of EGTA, N- and C-terminal halves G1–G3 and G4–G6 showed reduced binding with Kd = 0.495 ± 0.178 and
1.312 ± 0.256 μM, respectively, suggesting that binding of G4–G6 to NMMIIA requires calcium. (J) Pull-down assays
show pellets in Coomassie-stained gels. GST-Sepharose bead–bound gelsolin did not bind to HMM IIA (1–1337) in the
presence or absence of EGTA. (K) NMMIIA-His-thioredoxin residues 1339–1891 associated with full-length gelsolin
G1-G6–Sepharose beads in absence of EGTA. (L) NMMIIA residues 1899–1960 bound to GST-Sepharose beads did not
bind to gelsolin in the presence or absence of EGTA.
738 | P. D. Arora et al. Molecular Biology of the Cell
(Figure 3A). Collectively these data indicate that gelsolin/NMMIIA
complexes had no effect on the polymerization or capping activity of
gelsolin, but that binding of NMMIIA to gelsolin minimally reduced
the severing activity of gelsolin. In experiments of similar design,
when the rod domain of NMMIIA (residues 1339–1891) was incu-
bated with gelsolin, there was also reduced severing of actin fila-
ments compared with severing by gelsolin alone (Figure 3C). In con-
trast, when NMMIIA residues 1–1338 (Figure 3B) or NMMIIA residues
1899–1960 (Figure 3D) were incubated with gelsolin, there was no
effect on actin severing.
Gelsolin’s severing activity in cells
Because our in vitro data showed that gelsolin severing may be af-
fected by binding to NMMIIA, we determined whether gelsolin sev-
ering in intact cells is also affected by NMMIIA. We performed sev-
ering assays on lysates prepared from cells that had been plated for
1 h on planar collagen (actively spreading cells [S]) or after overnight
incubation on collagen (quiescent cells [Q]). The severing activity of
cells was slightly (30%) lower in cells plated overnight on collagen
(quiescent) compared with actively spreading cells plated for 1 h on
collagen (p < 0.1; Figure 3E).
We transfected wild-type and gelsolin-null cells with NMMIIA
small interfering RNA (siRNA) or a control siRNA and examined
whether NMMIIA knockdown affected the severing activity of wild-
type and gelsolin-null cells. The protein expression levels of
NMMIIA were similar in gelsolin-null and WT cells (Figure 3F). Cells
transfected with NMMIIA siRNA showed 75% reduction of NMMIIA
expression levels, but gelsolin protein levels in these cells were
unchanged (Figure 3G).
In quiescent cells (overnight plating on collagen), the severing
activity of the cell lysates was ∼50% lower in the gelsolin-null cells
compared with wild-type cells (Figure 3H), indicating that about one-
half of the actin-severing activity in these cells is attributable to gelso-
lin. NMMIIA knockdown did not affect the severing activity in actively
spreading gelsolin-null cells compared with gelsolin-null cells trans-
fected with control siRNA (Figure 3H). In gelsolin wild-type cells after
NMMIIA knockdown there was a small increase of severing activity
(by ∼25%; p < 0.1) compared with control cells (Figure 3H). Therefore
in both intact cells and in assays of purified proteins, NMMIIA ex-
erted a small inhibitory effect on gelsolin-mediated actin severing.
Collagen phagocytosis on planar and beaded surfaces
Because gelsolin and NMMIIA did not localize markedly to adhesion
complexes on planar surfaces but are known to be important for
phagocytosis of collagen-coated beads (Arora et al., 2004, 2008b),
we examined the effect of substrate topography on collagen degra-
dation. We analyzed the relative abundance of fluorescein isothio-
cyanate (FITC)–collagen in four cellular compartments that are asso-
ciated with collagen degradation in cells plated on planar surfaces or
on 2-μm-diameter latex beads (Figure 4A). We first determined the
total area and relative abundance of FITC-collagen coated on planar
surfaces (47.1 μg/cm2) and on 2-μm beads (49.6 μg/cm2), indicating
that the abundance of collagen on these different surfaces was very
similar. In cells plated on planar surfaces, ∼10% of the substrate col-
lagen was degraded and released into the medium, ∼35% of colla-
gen was associated with the cell surface, <10% was internalized, and
45% was not degraded (i.e., remained attached to the substrate;
Figure 4A). For cells plated on 2-μm collagen-coated beads, 18% of
collagen was degraded and released into the medium, 22% was as-
sociated with the cell surface, 45% was inter nalized, and 15% was
not degraded (Figure 4A). Given that the percentage of internalized
collagen was approximately fourfold higher in cells plated on beaded
constant Kd = 0.158 ± 0.082 μM; Figure 2D). In the presence of EGTA
the binding was reduced (Kd = 0.423 ± 0.152 μM; Figure 2E). The N-
and C-terminal halves of gelsolin (G1–G3, G4–G6) showed binding
with Kd = 0.220 ± 0.049 and 0.212 ± 0.063 μM, respectively (Figure 2,
F and G). In the presence of EGTA, the N- and C-terminal halves of
gelsolin (G1–G3, G4–G6) showed reduced binding, with Kd = 0.495 ±
0.178 and 1.312 ± 0.256 μM, respectively, suggesting that binding of
G4–G6 to NMMIIA requires calcium ions (Figure 2, H and I).
Mapping of binding domains in gelsolin
and NMMIIA filaments
With pull-down assays we examined the binding of truncated forms
of NMMIIA to full-length gelsolin (G1–G6). In pull-down assays,
gelsolin bound to GST-Sepharose beads did not associate with resi-
dues 1–1338 of NMMIIA (heavy meromyosin [HMM]) in the pres-
ence or absence of EGTA (Figure 2J). GST-Sepharose bead–bound
gelsolin cosedimented with NMMIIA–histidine (His)-thioredoxin
residues 1339–1891 in the presence of calcium ions (Figure 2K).
GST-Sepharose beads bound with the tail region of NMMIIA (resi-
dues 1899–1960) and that were incubated with full-length gelsolin
did not sediment gelsolin in the presence or absence of EGTA
(Figure 2L). Taken together, these data suggest that the rod region
enables binding of NMMIIA to gelsolin. In a control experiment pu-
rified GST did not cosediment with full-length NMMIIA filaments,
indicating specific binding of gelsolin to NMMIIA (Supplemental
Figure 1F). Similarly, gelsolin bound to GST-Sepharose did not
cosediment with His-thioredoxin (Supplemental Figure 1G).
Effect of NMMIIA on gelsolin-induced actin polymerization
We determined whether the interaction of gelsolin with NMMIIA
affects the nucleating, capping, and severing functions of gelsolin
(Yin, 1987). To assess the effects of gelsolin on actin polymerization,
we used a pyrene-labeled actin assay. The addition of gelsolin to
actin monomers sharply increased the rate of pyrene fluorescence,
but the addition of gelsolin/NMMIIA complexes at various NMMIIA
concentrations to pyrene–actin monomers exerted no effect on the
actin nucleation function of gelsolin (Supplemental Figure 2A).
NMMIIA alone exerted no effect on actin polymerization (Supple-
mental Figure 2B).
Effect of NMMIIA on gelsolin’s capping activity
We measured the capping activity of gelsolin/NMMIIA complexes
by incubating this complex with pyrene actin monomers in polymer-
ization buffer for 18 h. In the presence of gelsolin/NMMIIA com-
plexes, the final pyrene–actin fluorescence was similar for both
gelsolin alone and the gelsolin/NMMIIA complexes (Supplemental
Figure 2C). The addition of NMMIIA alone had no effect on pyrene–
actin fluorescence (Supplemental Figure 2D). These data indicate
that when NMMIIA is bound to gelsolin, there is no effect of
NMMIIA on the actin-capping activity of gelsolin.
Effect of NMMIIA on gelsolin’s severing activity
We investigated the effect of purified gelsolin/NMMIIA complexes
on actin filament severing by gelsolin. Full-length NMMIIA and gelso-
lin were coincubated and were added to pyrene-labeled actin fila-
ments; the rate of fluorescence loss was compared with the addition
of gelsolin alone. When gelsolin was added to pyrene-labeled actin
filaments, there was an initial loss of fluorescence due to severing of
actin filaments by gelsolin. When various concentrations of the gelso-
lin/NMMIIA complex were added to pyrene-labeled actin filaments,
there were small reductions of F-actin severing, which was slightly
lower (30% reduction) than the severing generated by gelsolin alone
Volume 24 March 15, 2013 Collagen adhesions | 739
FIGURE 3: (A) Histogram shows loss of fluorescence of pyrene-labeled actin filaments (0.3 μM) as a result of severing by
gelsolin (0.1 μM) in the presence of 1 mM CaCl2. Addition of purified NMMIIA (0.01, 0.02, 0.05, 0.1, 0.2, and 0.4 μM)
and gelsolin (0.1 μM) complex show decreased reduction of fluorescence compared with gelsolin alone. (B–D) In
experiments of similar design, NMMIIA domains consisting of residues 1–1338, 1339–1891, or 1899–1960 were
incubated with gelsolin, and actin severing was measured. (E) Lysates prepared from cells spreading on collagen-coated
beads show increase in severing of pyrene-labeled actin filaments compared with cells grown overnight on collagen-
coated tissue culture plates. Inset, G-purified gelsolin standard; S, Q, gelsolin expression levels in cell lysates prepared
from spreading (S) and quiescent (Q) cells. (F) Total cell lysates from gelsolin-null and wild-type cells were
immunoblotted for the indicated proteins. (G) Cells transfected with NMMIIA siRNA show 75% reduction in NMMIIA
protein levels compared with cells treated with nontargeted siRNA. (H) Wild-type or gelsolin-null cells transfected with
control siRNA or NMMIIA siRNA were maintained in quiescence or were spread on collagen. Cell lysates were analyzed
for actin severing by loss of pyrene fluorescence. Wild-type cells transfected with NMMIIA siRNA show enhanced
severing of actin filaments in actively spreading cells compared with cells transfected with irrelevant, control siRNA.
740 | P. D. Arora et al. Molecular Biology of the Cell
Further, in gelsolin-null cells or cells with
NMMIIA knockdown, there was reduced
collagen internalization only in cells plated
on beaded substrates and not on planar
substrates. These data suggested that
gelsolin and NMMIIA are important proteins
in mediating collagen internalization on
topologically complex surfaces.
Because collagen internalization was in-
hibited in gelsolin-null cells, we examined
whether collagen proteolysis in intracellular
compartments was different in gelsolin-null
cells compared with gelsolin wild-type cells.
Cells were plated on beads coated with ex-
ogenous biotinylated collagen for 2 or 8 h
and detached with trypsin from the plates,
and exogenous collagen that had been inter-
nalized was probed in cell lysates with strepta-
vidin peroxidase (Supplemental Figure 3C).
These data showed markedly more collagen
degradation in cells expressing gelsolin than
in gelsolin-null cells. Of note, the vacuolar
hydrolases that cleave collagen in the phago-
cytic pathway produce multiple collagen
cleavage fragments of widely varying mole-
cular mass, in contrast to the interstitial collagenases, which typically
degrade collagen into one-fourth and three-fourths fragments.
Ca2+ mobilization in response to surface topology
The importance of gelsolin and NMMIIA in collagen internalization
and the requirement for increased Ca2+ for gelsolin-NMMIIA interac-
tions motivated us to determine whether substrate topology affects
intracellular calcium responses during cell spreading. In cells loaded
with Fura-2/AM and plated on collagen-coated beads, there were
large increases of free intracellular calcium ion concentration ([Ca2+]i)
over time. The increase in [Ca2+]i was fourfold higher (Figure 5A) in
cells plated on collagen-coated beads than in cells plated on planar
collagen-coated surfaces (Figure 5D). We considered that cells very
likely undergo membrane stretch during spreading over collagen-
coated beads but less so on planar surfaces. Accordingly, we found
that treatment of cells with the stretch-activated channel inhibitor
Grammostola spatulata mechanotoxin 4 (GsMTx-4; 10 μM), reduced
collagen-induced increases of [Ca2+]i more markedly in cells plated
on beaded surfaces than on planar surfaces (Figure 5, B and E). In
cells incubated in an EGTA-containing buffer, there was minimal in-
crease of [Ca2+]i in cells plated on collagen beads or collagen-coated
planar surfaces (Figure 5, C and F). Similarly, chelation of intracellular
Ca2+ with BAPTA/AM reduced Ca2+ transients in these cells (Figure
5, C and F), suggesting that Ca2+ flux through stretch-activated
channels might be in part responsible for the Ca2+ transients exhib-
ited by cells spreading on beaded collagen surfaces.
We determined whether the effect of GsMTx-4 on [Ca2+]i was
restricted to entry of Ca2+ through stretch-activated channels, since
there is the possibility that GsMTx-4 might inhibit store-operated
Ca2+-permeable channels in the plasma membrane (Wang et al.,
2001). First we depleted cytoplasmic Ca2+ by incubation of cells in
buffer with very low Ca2+, followed by thapsigargin treatment, and
then we activated store-operated channels by addition of extracel-
lular Ca2+ in the presence or absence of GsMTx-4. These experi-
ments showed no inhibition of Ca2+ entry (Figure 5, G and H), indi-
cating that GsMTx-4 did not cause global inactivation of plasma
membrane Ca2+ channels.
compared with planar substrates (p < 0.001), substrate topography
evidently affects collagen internalization.
We performed similar experiments at 4ºC and observed greatly
decreased collagen degradation in cells plated on planar or beaded
surfaces (Figure 4B). Inhibition of collagenase activity with N-hydroxy-
carboxamide, an MMP-1 inhibitor (Pikul et al., 1998), reduced col-
lagen internalization to low levels for cells plated on collagen-coated
planar or beaded surfaces. In these experiments most of the colla-
gen was either bound to the cell surface or was not degraded
As shown earlier, in cells plated on planar collagen substrates,
collagen internalization was minimal, and gelsolin and NMMIIA did
not colocalize with vinculin-stained adhesions (Figure 1). We deter-
mined whether cells plated on larger collagen-coated beads (2, 20,
and 45 μm; Supplemental Figure 3A) would exhibit collagen inter-
nalization when the beads were sufficiently large that they could not
be internalized. We found that cells adhered to all sizes of beads but
did not ingest the larger beads (20 and 45 μm), as determined by
trypan blue quenching of FITC-collagen on the bead surface (Arora
et al., 2008a; unpublished data). Of note, for cells plated on 20- or
45-μm collagen-coated beads, the compartmentalization of colla-
gen degradation was similar to the pattern observed in cells that
were plated on planar surfaces; in these preparations there was
minimal internalized collagen, as measured with FITC-collagen
(<4%; Supplemental Figure 3B).
Because the immunostaining data (Figure 1) suggested that
gelsolin and NMMIIA localize to adhesions around collagen beads
but not on planar substrates, we studied the role of gelsolin and
NMMIIA in collagen remodeling in cells plated on the same planar
or beaded collagen substrates. Gelsolin-null cells or wild-type cells
or gelsolin wild-type cells transfected with NMMIIA siRNA were
plated on FITC-collagen–coated planar surfaces or FITC-collagen–
coated beaded (2 μm) surfaces. Compared with wild-type cells,
there was >10-fold reduction in collagen internalization for gelsolin-
null cells or cells treated with NMMIIA siRNA (Figure 4, D and E), but
there was no difference of collagen release into the medium.
FIGURE 4: (A) Comparison of collagen degradation on collagen-coated planar surface and
collagen-coated 2-μm beads. Histogram shows FITC-collagen associated with discrete cellular
compartments. The data are percentage of FITC-collagen released into media, cell-surface
bound, internalized, or remaining (nondegraded) adherent to substrate (planar or bead
surfaces). (B, C) Collagen internalization was retarded at low temperatures and in the presence
of MMP inhibitor. (D, E) Histograms show collagen on planar or irregular surface metabolized by
gelsolin-null, WT cells transfected with myosin IIA siRNA.
Volume 24 March 15, 2013 Collagen adhesions | 741
positively stained regions of interest around collagen beads within
2 min after bead incubation, which preceded the appearance of posi-
tive immunostaining for gelsolin (not seen until 5–15 min). Regions
of interest that were stained for gelsolin were always positive for
NMMIIA. Thus NMMIIA might function in the recruitment of gelsolin
to sites of bead adhesion (Figure 6A): <1% of gelsolin-positive re-
gions of interest did not show corresponding NMMIIA fluorescence.
The percentage of positively stained regions of interest for
NMMIIA and gelsolin increased over time after plating, and this pro-
cess was strongly dependent on the presence of extracellular Ca2+,
since chelation of extracellular Ca2+ (1 mM EGTA-containing buffer)
reduced gelsolin recruitment (Figure 6B). Compared with gelsolin,
NMMIIA- and calcium-dependent enrichment of adhesion
sites with gelsolin
Because immunostaining showed that gelsolin and NMMIIA were
localized to collagen-bead adhesion sites (Figure 1), we determined
the temporal sequence of the enrichment of these proteins at
collagen-bead adhesions. Wild-type fibroblasts were incubated with
collagen-coated beads, fixed after discrete time points, and immu-
nostained for gelsolin and NMMIIA. Fluorescence intensity was mea-
sured in regions of interest around beads that exhibited staining in-
tensities above background thresholds for gelsolin and NMMIIA;
these data were scored as percentage of positive regions of interest
(50 cells in each experimental condition). There were 25% NMMIIA
FIGURE 5: (A, D) Fura-2/AM–loaded cells plated on collagen-coated beads or collagen-coated planar surface show
increase in intracellular calcium levels ([Ca2+]i) over time. Images show estimates of intracellular calcium levels in
pseudocolor. (B, E) Treatment of cells with the stretch-activated channel (SAC) inhibitor GsMTx-4 (10 μM) reduced [Ca2+]i
increases in response to plating on collagen. (C, F) In the presence of EGTA-containing buffer, the increase in [Ca2+]i was
minimal in cells plated on collagen beads or collagen-coated planar surface. Chelation of intracellular Ca2+ with BAPTA/
AM also reduced Ca2+ transients. (G, H) Lack of effect of GsMTx-4 on store-operated channels. Cells were subjected to
store depletion of intracellular calcium stores with low Ca2+ buffer, followed by thapsigargin (1 μM), and treated with
either vehicle (G) or GsMTx-4 (10 μM; H).
742 | P. D. Arora et al. Molecular Biology of the Cell
(Gsn 100/210; Arora et al., 2005) that exhibits marked reduction of
actin severing compared with wild-type gelsolin, as measured by
fluorescence loss of pyrene-labeled actin filaments (Figure 6D). In
vitro pelleting assays showed association of full-length NMMIIA
with the gelsolin severing mutant (Gsn 100/210) in calcium-
containing buffer (Figure 6E). Further, in gelsolin-null cells trans-
fected with the severing mutant, although there were reduced
numbers of bound collagen beads, gelsolin immunostaining colo-
calized with NMMIIA at collagen bead–binding sites (Figure 6F).
We examined the collagen degradation patterns in cells plated
on planar or beaded substrates in gelsolin-null cells that were trans-
fected with a wild-type gelsolin expression construct or in gelsolin-
null cells transfected with the gelsolin severing mutant (Gsn 100/210).
In gelsolin-null cells transfected with cDNA for wild-type gelsolin
there was a >10-fold increase in internalized collagen in cells plated
the percentage of positively stained regions for NMMIIA was much
less affected by the chelation of Ca2+. Of note, pretreatment of cells
with GsMTx-4 (10 μM) blocked colocalization of NMMIIA and gelso-
lin at bead sites (Figure 6C), indicating that Ca2+ transients are likely
important for association of NMMIIA and gelsolin at collagen adhe-
sion sites. We also examined the potential functional significance of
NMMIIA activity by treating cells with blebbistatin (50 μM) but found
that targeting of NMMIIA and gelsolin to collagen-coated beads
was not affected by blebbistatin (Supplemental Figure 3D).
Role of gelsolin and NMMIIA in collagen degradation
Because NMMIIA had no effect on gelsolin polymerization and
capping functions, we examined whether the severing activity of
gelsolin was important for collagen degradation mediated by
the intracellular pathway. We used a gelsolin severing mutant
FIGURE 6: (A, B) Quantitative analysis of localized fluorescent regions of interest around bead attachment sites. The
data show the percentage of positively stained regions of interest around bound collagen beads for cells
immunostained with gelsolin and NMMIIA. Data were collected over time in the presence or absence of EGTA.
(C) Pretreatment of cells with GsMTx-4 (10 μM) blocked colocalization of NMMIIA and gelsolin at bead sites. (D) Loss of
fluorescence of pyrene-labeled actin filaments (0.3 μM) as a result of severing by wild-type gelsolin (0.1 μM) or the
severing mutant (Gsn 100/210). (E) Pelleting assays show association of full-length NMMIIA with gelsolin severing
mutant (Gsn 100/210) in the presence or absence of EGTA in a Coomassie-stained, SDS-polyacrylamide gel. (F) Gelsolin
null cells transfected with gelsolin cDNA and severing mutant showing localization of gelsolin and NMMIIA to collagen
beads. (G, H) Histogram showing requirement of severing activity of gelsolin for collagen internalization during
compartmentalization of FITC-collagen coated on beads. Severing mutant (Gsn 100/210) shows retarded collagen
internalization. (I) Stretch-activated channel inhibitor (GsMTx-4) prevents internalization and collagen degradation.
Volume 24 March 15, 2013 Collagen adhesions | 743
Cell-spreading processes on flat surfaces are fundamentally
similar to the behavior of cells plated on very large fibronectin-
coated beads (Grinnell and Geiger, 1986). We observed that col-
lagen degradation was affected by the nature of the adhesions
(planar or irregular surfaces), which also determined the recruit-
ment of adhesion-regulating proteins that are required for remod-
eling by phagocytosis (Everts et al., 1996). The gene product of the
Drosophila lethal giant larvae regulates NMMIIA cellular distribu-
tion and focal adhesion morphology to optimize cell migration
(Dahan et al., 2011), which suggests that NMMIIA might play a
fundamental role in determining adhesion function in different cells
and cellular processes.
We found that collagen binding increased [Ca2+]i, which might
reflect activation of stretch-activated calcium channels. Regulation of
cell movement is also mediated by stretch-activated calcium channels
(Lee et al., 1999). Our studies of purified proteins showed that gelso-
lin and NMMIIA interactions are Ca2+ dependent. Ca2+ also enhances
the actin-severing function of gelsolin (Kiselar et al., 2003), which in
turn is important for initiating actin remodeling at the cytoplasmic
side of collagen-binding sites (Arora et al., 2011). Therefore the col-
lagen bead–evoked Ca2+ that occur early on in the binding process
might play a fundamental role in enabling subsequent recruitment of
critical actin-modifying proteins to the nascent phagosome.
We identified residues 1339–1891 of the NMMIIA heavy chain as
a gelsolin-interacting domain, a region that is also important for
NMMIIA filament assembly (Dulyaninova et al., 2005). Examination
of gelsolin and NMMIIA interacting domains indicated that gelsolin
domains 4 and 6 interact with NMMIIA in a Ca2+-dependent man-
ner. Gelsolin adopts an activated state in the presence of Ca2+ in
which three masked actin-binding sites are exposed; these include
actin monomer–binding sites on domains G1 and G4, and a fila-
ment side–binding site on G2 (Way et al., 1989; Pope et al., 1991).
However, crystallization studies of the C-terminal half of gelsolin in
the presence of Ca2+ (but in the absence of actin) demonstrated that
actin is not a prerequisite for enabling the development of an acti-
vated gelsolin conformation (Narayan et al., 2003). Our data show
that, unlike the N-terminal half of gelsolin (domains G1–G3), the
C-terminal half of gelsolin (G4–G6) does not interact with NMMIIA
in the presence of EGTA. It is conceivable that the interaction be-
tween NMMIIA and gelsolin domains 4 and 6 requires Ca2+ to en-
able conformational changes that are consistent with the interaction
of the two molecules.
Effect of NMMIIA on gelsolin function
Actin capping, nucleation, and severing are important functions of
gelsolin required for regulation of adhesion maturation and colla-
gen matrix remodeling. We considered that the interaction of
NMMIIA with gelsolin might affect gelsolin function. Our in vitro
data indicated that when NMMIIA was bound to gelsolin, there was
no effect on gelsolin-mediated capping or nucleation function and
only a small inhibition of gelsolin’s severing activity. It is conceivable
that a small inhibition of the severing function of gelsolin by binding
to NMMIIA might be necessary initially to prevent actin depolymer-
ization in the locale of adhesions. Another, more likely possibility is
that since NMMIIA sequesters gelsolin to collagen bead–binding
sites, small reductions of gelsolin’s severing activity are more than
made up for by the abundance of this abundant actin-remodeling
protein at the nascent phagosome, where it enables actin polymer-
ization and capping (Arora et al., 2011). Indeed, our experiments on
intact cells show that, whereas knockdown of NMMIIA inhibits total
on beaded collagen (but not planar substrates) compared with
gelsolin-null cells (Figure 4D). When gelsolin-null cells were trans-
fected with the gelsolin severing mutant construct (Arora et al.,
2005), collagen internalization and the amount of degraded colla-
gen were similar to that of gelsolin-null cells (Figure 6, G and H), and
there was no difference in the pattern of collagen degradation in
cells plated on beaded or planar substrates. Taken together, these
data indicate that gelsolin’s severing activity is important for colla-
gen phagocytosis, but this process operates only when collagen is
presented on a beaded surface.
As described here, we found that in cells binding to collagen
beads, there was a marked increase of [Ca2+]i that was inhibited by
treatment with the stretch-activated cation channel inhibitor
GsMTx-4 (Figure 5). Because Ca2+ was evidently crucial for bind-
ing of NMMIIA to gelsolin (Figure 2) and is known to be important
for gelsolin’s severing activity (Yin et al., 1981), we treated cells
plated on collagen with GsMTx-4 or vehicle and examined colla-
gen internalization. There was ninefold-reduced collagen internal-
ization in cells treated with GsMTx-4 compared with vehicle con-
trols (p < 0.001; Figure 6I).
The potential interactions involving gelsolin and NMMIIA that
may regulate early steps of collagen phagocytosis are not
defined. Because the flattened appearance of cultured fibroblasts
plated on planar surfaces differs from their in vivo appearance
(Grinnell, 2003), which tends to be of a more complex dendritic
shape, we examined whether collagen substrate topography
affects the recruitment of gelsolin and NMMIIA to the adhesions,
their interactions, and the nature of collagen degradation. We
found that gelsolin and NMMIIA are enriched in cell adhesions
that mediate collagen phagocytosis but only when cells are plated
on a beaded and not on planar collagen substrates. Further,
NMMIIA binds gelsolin at collagen adhesion sites; this Ca2+-
dependent interaction enables actin remodeling at adhesion sites
(Arora et al., 2011), which is required for collagen phagocytosis.
One of the regulatory systems that enable phagocytosis involves
activation of stretch-sensitive, Ca2+-permeable channels when
cells attach and stretch over small, collagen-coated beads. The
entry of Ca2+ through these channels may be stimulated by the
extensive shape changes of fibroblasts that are manifest when en-
gage collagen fibrils in vivo (ten Cate, 1972; Svoboda et al., 1979;
Melcher and Chan, 1981). Of note, humans and animals that are
treated with drugs that inhibit Ca2+ entry exhibit inhibition of the
collagen phagocytosis pathway and fibrosis (McCulloch and
Knowles, 1993; McCulloch, 2004).
NMMIIA and gelsolin at adhesion sites
We found that NMMIIA and gelsolin do not colocalize with vinculin-
stained focal adhesions in cells spread on planar surfaces but are
recruited to adhesions forming on beaded substrates, which may
more closely mimic in vivo conditions (Melcher and Chan, 1981).
NMMIIA is recruited to nascent adhesions in the lamella in migrat-
ing cells (Burnette et al., 2011), whereas NMMIIB associates with
actin filament bundles in stable focal adhesions (Vicente-Manzanares
et al., 2007). Of note, myosin VI plays specific roles in different intra-
cellular functions such as endocytosis and cell migration (Buss et al.,
2002), and myosin X associates with actin filaments at the distal tips
of filopodia involved in rapid back and forth oscillations during ret-
rograde flow of bundled actin (Nagy et al., 2008). It is conceivable
that NMMIIA may play specific roles in adhesion to and phagocyto-
sis of collagen in fibroblasts.
744 | P. D. Arora et al. Molecular Biology of the Cell
Calbiochem (San Diego, CA). FITC-labeled,
bovine type I collagen (5 mg/ml) was
purchased from AnaSpec (Fremont, CA).
Pyrene-labeled actin was from Cytoskeleton
(Denver, CO). Fura-2/AM was from Invitro-
gen (Mississauga, Canada). FFP-18 was from
Teflabs (Austin, TX). GsMTx-4, an inhibitor
of stretch channels, was from Alomone
Fibroblasts were derived from gelsolin wild-
type and null mice as described (Arora et al.,
2004). NMMIIA wild-type embryonic stem
cells and NMMIIA-null embryonic stem cells
were obtained from R. Adelstein (National
Institutes of Health, Bethesda, MD) and
cultured as described (Arora et al., 2008a).
Collagen bead binding and cell plating
Unlabeled collagen-coated latex beads
(2 μm diameter) were applied to microbio-
logical (i.e., non–tissue culture) dishes, dried
down, and attached as described (Arora
et al., 2003), followed by washing with phos-
phate-buffered saline (PBS). The number of
beads plated per dish was adjusted to pro-
duce final bead:cell ratios specific for each
experiment. Cells were counted electroni-
cally, and the cell concentration was ad-
justed before plating cells on dishes con-
taining collagen-coated beads. The plates
were maintained at room temperature for
20 min to allow the cells to settle and subse-
quently washed with fresh medium at 37°C.
Detached cells were removed by repeated
washes. In experiments that evaluated sites
of collagen degradation (see later descrip-
tion), the amount of collagen bound to
beads or to plates was determined using
FITC-collagen, fluorimetry, and estimates of the surface areas of ei-
ther the planar substrates or the beaded substrates that were used
in specific experiments.
We determined collagen degradation in cells plated on collagen
substrates with different topologies. For planar substrates, 48-well
cell culture plates were coated with FITC-collagen (30 min). Plates
were allowed to dry after removing excess, unbound collagen solu-
tion; nonspecific combining sites were blocked with 1% bovine se-
rum albumin (BSA) solution. For beaded substrates, latex beads
(2, 20, and 45 μm diameter) were coated with FITC-collagen, plated
in the wells, allowed to settle for 2 h, and dried after aspiration of
excess fluid, and nonspecific combining sites were blocked with
BSA. We measured the amounts of FITC-collagen attached to the
respective substrates by treatment with bacterial collagenase, fol-
lowed by photon counting of the released FITC-collagen. The
amounts of collagen on the various substrates were adjusted so
that each surface exhibited equivalent collagen loading for equiva-
lent surface areas.
Cells (5 × 104) were plated in each well and incubated for 1 h.
After cell incubations, the medium in each well contained released
cellular actin-severing activity, this same type of knockdown mark-
edly inhibits collagen internalization.
In summary, cell adhesion to topologically complex surfaces such
as collagen beads activates Ca2+ entry through stretch-activated
channels and promotes the formation of phagosomes enriched with
NMMIIA and gelsolin. The Ca2+-dependent interactions of gelsolin
with NMMIIA enable actin remodeling at collagen bead adhesion
sites, which enhances collagen degradation by the phagocytic path-
way (Figure 7).
MATERIALS AND METHODS
Latex beads (2, 20, and 45 μm in diameter) were purchased from
Polysciences (Warrington, PA). Purified, pepsin-digested, bovine
type I collagen was purchased from Advanced BioMatrix (San Diego,
CA). Antibodies to β-actin (clone AC-15), myosin light chain, glycer-
aldehyde-3-phosphate dehydrogenase, FITC-conjugated goat
anti-mouse antibody, and tetra-methyl rhodamine isothiocyanate–
phalloidin were obtained from Sigma-Aldrich (Oakville, Canada).
Clostridial collagenase was from Sigma-Aldrich. MMP inhibitor II (N-
erazine-2-carboxamide) and blebbistatin were obtained from
FIGURE 7: Schematic model of calcium regulation of gelsolin interaction with NMMIIA. Cell
adhesion to topologically complex collagen substrate induces [Ca2+]i flux, which promotes
interaction of NMMIIA rods with gelsolin domains G1–G3 and G4–G6. This interaction enhances
actin remodeling by gelsolin at collagen adhesion sites to enable collagen phagocytosis. Low
[Ca2+]i inhibits binding of NMMIIA with gelsolin domains G4–G6, which results in reduced actin
remodeling and inhibition of collagen phagocytosis.
Volume 24 March 15, 2013 Collagen adhesions | 745
The GST-gelsolin C-terminal half (G4–G6) construct was provided
by H. L Yin. Briefly, the gene fragment coding for residues 414–746
of gelsolin (domains G4–G6) was ligated into a modified PGEX-6P-1
plasmid (Amersham) using the PCR (Narayan et al., 2003). Domains
G4–G6 were expressed in E. coli XL-1 Blue cells grown in Luria broth
(LB) containing 100 μg/ml ampicillin and induced with 0.5 mM
isopropyl-β-d-thiogalactoside (IPTG)for 3 h at 30ºC.
Purified HMM of NMMIIA (1–1339) was provided by J. Sellers
(National Institutes of Health, Bethesda, MD). GST-tagged 1899–
1960 NMMIIA was expressed and purified as described previously
(Li et al., 2003).
To prepare the myosin construct consisting of amino acids 1339–
1891, a primer pair (forward, 5′-GCGCGAATTCAATTCCTTCCGG-
GAGCAGCT-3′; reverse, 5′-GCGCGCGGCCGCACTAGGCGTTG-
GCCCGCTGG-3′) was designed according to GenBank number
P35579.4 to generate EcoRI and NotI restriction sites (underlined)
that flanked human NMMIIA (amino acids 1339–1891). We used
pET-28a (+)-hNMMIIA 1339-end from one of us (A.B.) as the tem-
plate. The resulting PCR product was ligated into pET-32a (+) (Nova-
gen, Gibbstown, NJ; provided by Y. Yao and M. F. Manolson,
University of Toronto, Toronto, Canada) digested by EcoRI and NotI.
The sequence of the insert was confirmed by sequencing (ACGT).
This construct was transformed into BL21 (DE3) cells for protein ex-
pression and purification. His-thioredoxin expressed by the original
vector in BL21 (DE3) was used as the control. Nickel–nitriloacetic
acid superflow columns (Qiagen, Valencia, CA) and nondenaturing
conditions were used for protein purification.
GST-tagged proteins expressed in bacterial expression systems
were isolated and purified as described previously (Puius et al.,
2000; Arora et al., 2005). Briefly, for production of gelsolin proteins,
BL21(DE3) cells were transformed with wild-type constructs. Luria
broth (250 ml) containing ampicillin (100 μg/ml) was inoculated
overnight at 37ºC, followed by induction with IPTG (1 mM) for 4 h.
Equivalent expression levels of the mutants was examined by im-
munoblotting. Proteins isolated from inclusion bodies were dialyzed
overnight and loaded onto glutathione–Sepharose 4B columns
(Pharmacia, Sigma-Aldrich, Oakville, ON, Canada). The fusion pro-
teins were cleaved on the column with thrombin (20 U in 2 ml of
PBS) after overnight incubation. The eluate containing gelsolin and
thrombin was separated by Centricon 50 filters (Amicon, Millipore,
Billerica, MA). Protein yields were determined by optical density and
Purification of full-length nonmuscle myosin
Full-length NMMIIA was isolated from J744 macrophages as de-
scribed (Trotter and Adelstein, 1979). Briefly, cells (107) were washed,
collected by low-speed centrifugation, and resuspended in ice-cold
extraction buffer (340 mM sucrose, 15 mM Tris-HCl, pH 7.5, 1 mM
EDTA, 10 mM dithiothreitol [DTT], and 10 mM Na4P2O7 plus pro-
tease inhibitors) equivalent to two times the weight of the pellet.
The cell suspension was stirred for 45 min in the cold, and cells were
broken in a Dounce homogenizer. The volume of the cell suspen-
sion was brought to three times the weight of the pellet. The homo-
genate was centrifuged at 40,000 × g for 40 min at 4ºC. The cell
extract was equilibrated to 10 mM in MgATP and then brought to
35% saturation by slow addition of saturated ammonium sulfate
containing 10 mM EDTA, pH 8.2. The precipitate was collected by
centrifugation at 15,000 × g for 30 min at 4ºC; the supernatant was
brought to 55% saturation in ammonium sulfate. The precipitated
proteins (35–55%) were collected by centrifugation and dissolved in
10 ml of high–ionic strength buffer (0.6 M KCl, 15 mM Tris HCl,
pH 7.5, 1 mM EDTA, 5 mM DTT). The solution was centrifuged, and
(i.e., extracellularly degraded) collagen that was collected for
analysis. After collection of the medium, PBS (200 μl × 3) was used
to wash each well, and washes were collected for further analysis.
These washes contained FITC-collagen that was loosely associ-
ated with the cell surface and was removed by washing. Trypsin
(1%; 200 μl) was used to detach cells from planar or beaded sub-
strates, and the FITC-collagen in these washes was added to the
previously described cell surface–associated collagen. Of note,
trypsinization did not remove beads from the bottom of the wells,
as assessed by microscopy of the trypsin fraction. The detached
cells in the trypsin fraction were centrifuged (5 min at 14,000 × g)
and then lysed in Triton-X (1%) buffer; this fraction contained
FITC-collagen that had been internalized by cells. Finally, bacte-
rial collagenase (1%; 200 μl) was added to solubilize the remain-
ing collagen attached to the bottom of the wells or to the bead
surfaces (nondegraded collagen fraction). All fractions were mea-
sured separately in a spectrofluorimeter (absorbance/emission =
492/520 nm; PTI, London, Canada). For each of the different types
of substrates, FITC-collagen photon counts were determined on a
percentage basis for each of the released, cell-surface, internal-
ized, and nondegraded fractions. In some experiments, a collage-
nase inhibitor (N-hydroxy-1,3-di-(4-methoxybenzenesulfonyl)-5,5-
dimethyl-[1,3]-piperazine-2-carboxamide) was used at 1 μM. This
inhibitor potently blocks MMP-1, with IC50 = 24 nM (Pikul et al.,
Gelsolin and nonmuscle myosin constructs
Human full-length gelsolin cDNA (Kwiatkowski et al., 1986) was pro-
vided by H. L. Yin (University of Texas, Dallas, TX). A primer (Gen-
Bank number X04412.1) pair (forward, 5′-GCGCAAGCTTGTGGTG-
GAACACCCCGAGTTCCTCAAGGCA-3′; reverse, 5′-GCGCGGATC-
CCTAAGACCAGTAATCATCATCCCAGCCAAG-3′) was designed to
amplify full-length gelsolin (G1–G6, 2.151 kb, encoding gelsolin
residues 26–742). A primer pair (forward, 5′-GCGCAAGCTTGTG-
GTGGAACACCCCGAGTTCCTCAAGGCA-3′; reverse, 5′-GCGC-
GGATCCCTAGTCCCGCCAGTTCTTGAAGAACTGCTT-3′) was used
to amplify the N-terminal three domains (G1–G3, 1.038 kb, encod-
ing gelsolin residues 26–371; Burtnick et al., 2004) and a primer pair
GATCTGG-3′; reverse, 5′-GCGCGGATCCCTAAGACCAGTAAT-
CATCATCCCAGCCAAG-3′) was used to amplify the C-terminal
three domains (G4–G6, 0.987 kb, encoding gelsolin residues 414–
742). The resulting PCR products were ligated into the HindIII/BamHI
sites (underlined sequences in the primers) of N3×FLAG-pCMV5c
(constructed by Miriam Barrios-Rodiles, Samuel Lunenfeld Research
Institute, Toronto, Canada) and sequenced (ACGT Corp., Toronto,
To prepare the GST-tagged terminal gelsolin half (GST-G1-3) con-
struct, a primer pair (forward, 5′-GCGCGAATTCAAGTGGTGGAA-
CACCCCGAGTTCCTCAAGG-3′; reverse, 5′-GCGCCTCGAGAC-
TAGTCCCGCCAGTTCTTGAAGAACTGCT-3′) was designed to
amplify the N-terminal three domains (G1–G3, 1.038 kb, encoding
gelsolin residues 26–371) of human gelsolin cDNA, flanked by
EcoRI and XhoI restriction sites (underlined). The resulting PCR
product was ligated into the corresponding sites of pGEX-4T-2
(Amersham, Oakville, Canada) and transformed into DH5α-
competent Escherichia coli cells (Invitrogen, Burlington, Canada).
The construct was sequenced (ACGT Corp.) and transformed into
BL21(DE3)-competent E. coli cells for GST-tagged protein expres-
sion and purification.
746 | P. D. Arora et al. Molecular Biology of the Cell
incubated with collagen-coated beads, fixed with 3% formaldehyde
in PBS, permeabilized with 0.2% Triton X-100, and immunostained.
The spatial distribution of staining around beads was determined by
confocal microscopy (Leica, Heidelberg, Germany; 40× oil immer-
sion lens). Transverse optical sections were obtained at 0.5-μm nom-
inal thickness using Leica software and analyzed with Photoshop
(Adobe, San Jose, CA). Colocalization was analyzed three to six
fields per cell depending, on the number of bound beads by using
the ImageJ plug-in (National Institutes of Health, Bethesda, MD;
Bolte and Cordelieres, 2006). At least 20 cells were examined for
each experimental condition. Pearson’s r of colocalizing proteins
was expressed as the mean ± SD. In some experiments we used
total internal reflection fluorescence (Leica) microscopy to selec-
tively illuminate fluorophores within ∼100 nm of the interface of
collagen bead attachments to the cell surface.
Intracellular Ca2+ measurements
Cells were loaded with Fura-2/AM (3 μM) or with FFP-18 (5 μM;
Teflabs, Austin, TX) and plated on collagen-coated beads or colla-
gen-coated planar substrates. Intracellular Ca2+ concentration
([Ca2+]i) in single, attached cells was estimated with a microscope-
based, ratio fluorimeter (Arora et al., 1994). In some experiments
that involved Ca2+ chelation, cells were treated with BAPTA/AM
(2 μM) for 30 min before experiments. In experiments to examine
the influence of stretch-sensitive channels on collagen-induced in-
creases of [Ca2+]i, GsMTx-4 (10 μM; Suchyna et al., 2000) was used.
For all continuous-variable data, means and SDs were computed.
When appropriate, comparisons between two samples were made
by Student’s t test with statistical significance set at p < 0.05. For
multiple comparisons, analysis of variance was used. All experiments
were performed at least three times in triplicate.
the clarified solution was dialyzed against low–ionic strength buffer
(60 mM KCl, 15 mM Tris HCl, pH 7.5, 1 mM EDTA, 5 mM DTT). The
precipitated protein was collected by centrifugation and dissolved
in 1–2 ml of high–ionic strength buffer. This crude fraction, desig-
nated as actomyosin, was made in 10 mM MgATP to dissociate actin
from myosin and clarified by centrifugation at 100,000 × g for
45 min. The clear solution was dialyzed to remove traces of MgATP.
To obtain a well-distributed suspension of myosin filaments, we re-
duced the salt concentration during the final dilution to 150 mM or
by dialysis into 150 mM KCl, 0.1 mM EGTA, 2 mM MgCl2, and
10 mM 3-(N-morpholino)propanesulfonic acid, pH 7.0.
In vitro binding assays
Protein concentrations were determined by running standards on
SDS polyacrylamide gels or by the bicinchoninic acid protein de-
termination method. To assess binding of NMMIIA, we dialyzed
rod filaments (residues 1338–1960) to GST–gelsolin full-length
(G1–G6), GST–gelsolin N-terminal half (G1–G3), or GST–gelsolin
C-terminal half (G4–G6) proteins against assembly buffer (20 mM
Tris, pH 7.5, 20 mM NaCl, 2 mM MgCl2, 1 mM DTT) overnight
before binding assays. Various concentrations of purified GST-
gelsolin proteins (0.25–6 μM) were incubated with assembled
NMMIIA rods (1 μM) at 23ºC for 45 min in the reaction buffer
containing 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 2 mM MgCl2,
0.3 mM CaCl2, and 1 mM DTT. Samples were centrifuged for
15 min at 80,000 rpm. Supernatant and pellet samples were sep-
arated by SDS–PAGE and stained with Coomassie blue. Similar
experiments were done with GST-gelsolin proteins bound to glu-
tathione–Sepharose in the presence of Ca2+ (0.3 mM) or in the
presence of EGTA (2 mM).
Actin-severing, -nucleation, and -capping assays
Lyophilized rabbit skeletal muscle actin or human platelet nonmus-
cle actin (both from Cytoskeleton) were resuspended in G-buffer
containing actin monomers (0.3 μM; 30% pyrenyl-G-actin) and was
polymerized in polymerization buffer for 2 h at room temperature.
The decrease in fluorescence due to actin severing by cell proteins
was measured in a fluorimeter (PTI; excitation, 365 nm; emission,
386 nm). For assessment of actin polymerization, lyophilized actin
(Cytoskeleton) was resuspended and dialyzed in G-buffer (2 mM Tris,
pH 8.0, 0.1 mM CaCl2, 0. 2 mM ATP, 0.5 mM β-mercaptoethanol)
overnight. G-actin (2 μM; 15% pyrenyl–G-actin) was treated with po-
lymerization buffer (25 mM Tris, pH 7.0, 50 mM KCl, 2 mM MgCl2,
0.1 mM ATP). The increase in fluorescence was measured in a PTI
fluorimeter (excitation, 365 nm; emission, 386 nm).
We measured the severing activity of lysates prepared from
gelsolin wild-type and null cells that were previously transfected
with NMMIIA siRNA or a control siRNA. Cell lysates were collected
with detergent plus protease inhibitors in buffer containing 50 mM
KCl, 2 mM MgCl2, 0.5 mM ATP, 2 mM Tris, pH 8.0, 1 mM EGTA, and
1% Triton X-100. Lysates were dialysed with several changes of buf-
fer containing 2 mM MgCl2, 50 mM KCl, 2 mM Tris-HCl, and 1 mM
EGTA and 0.5 mM β-mercaptoethanol. Purified gelsolin and gelso-
lin incubated with various concentrations of NMMIIA protein were
incubated with pyrene-labeled actin filaments in F-buffer (4 mM Tris,
pH 8.0, 50 mM KCl, 2 mM MgCl2, and 1 mM CaCl2), and the rate of
fluorescence loss was compared with that for gelsolin.
Immunofluorescence and confocal and total internal
reflection fluorescence microscopy
We determined the spatial distribution of endogenous gelsolin
and NMMIIA with respect to bound collagen beads. Cells were
Research was supported by a Canadian Institutes of Health Research
operating grant to C.A.M. (MOP-36332), whose salary is provided
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