Drosophila embryos close epithelial wounds using a combination of cellular protrusions and an actomyosin purse string

Article (PDF Available)inJournal of Cell Science 125(24) · October 2012with14 Reads
DOI: 10.1242/jcs.109066 · Source: PubMed
The repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular, which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process-expansion, coalescence, contraction, and closure-and describe the molecular dynamics of each phase. Specifically, we find that myosin, E-cadherin, Echinoid, the plasma membrane, microtubules, and the Cdc42 small GTPase respond dynamically during wound repair, and demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular protrusions and actomyosin ring contraction along the leading edge of the wound.
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Journal of Cell Science
Drosophila embryos close epithelial wounds using a
combination of cellular protrusions and an actomyosin
purse string
Maria Teresa Abreu-Blanco*, Jeffrey M. Verboon*, Raymond Liu, James J. Watts and Susan M. Parkhurst
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
*These authors contributed equally to this work
Author for correspondence (susanp@fhcrc.org)
Accepted 30 August 2013
Journal of Cell Science 125, 5984–5997
2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.109066
The repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular,
which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we
analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound
assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process,
expansion, coalescence, contraction and closure, and describe the molecular dynamics of each phase. Specifically, we find that myosin,
E-cadherin, Echinoid, the plasma membrane, microtubules and the Cdc42 small GTPase respond dynamically during wound repair. We
demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results
show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular
protrusions and actomyosin ring contraction along the leading edge of the wound.
Key words: Epithelial wound repair, Actomyosin ring, Cellular protrusions
Wound repair is an essential physiological process that is
compulsory for organism survival, tissue homeostasis, and to
avoid infections. From invertebrates to mammals, organisms
have developed robust and rapid responses to restore tissue
continuity during embryonic and adult life. An efficient wound
repair response is particularly important for tissues such as
the epithelia, which serves as protective barriers from the
surrounding environment and are constantly exposed to
chemical and mechanical trauma. Epithelial wound repair has
been studied in vivo in different model organisms and extensively
in vitro in cell/tissue culture (Martin and Lewis, 1992; Bement
et al., 1993; McCluskey et al., 1993; Brock et al., 1996; Danjo
and Gipson, 1998; Fenteany et al., 2000; Davidson et al., 2002;
Wood et al., 2002; Galko and Krasnow, 2004; Russo et al., 2005;
Tamada et al., 2007). From these studies, it has been proposed
that epithelial wounds heal through a variety of mechanisms,
including lamellipodial crawling/cell migration, contraction of
the cells underlying the wound, and actomyosin purse string-
mediated contraction of the cells at the wound periphery
(Fig. 1A) (Martin and Lewis, 1992; Bement et al., 1993; Brock
et al., 1996; Wood et al., 2002).
Several factors have been proposed to affect the choice of
repair mechanism to be employed, including the size and shape
of the wound, the intrinsic tension in the tissue, the nature of
junctions/connections between cells, and the developmental
stage. Re-epithelialization by lamellipodial crawling, where the
leading edge cells push themselves forward over the wound is
commonly observed in adult tissues (Fig. 1A) (Odland and Ross,
1968; Pang et al., 1978; Buck, 1979). Epithelial wound closure in
embryos is often mediated by the formation of a contractile
actomyosin purse string linked intercellularly by adherens
junctions (Martin and Lewis, 1992; Bement et al., 1993; Brock
et al., 1996) (Fig. 1A). However, the developmental stage of an
organism does not necessarily define the repair mechanism used,
as purse string-mediated repair has been observed in certain adult
tissues, including the adult cornea (Danjo and Gipson, 1998).
Some types of epithelial cells in culture repair their wounds using
a combination of purse string contraction and lamellipodial
crawling (Bement et al., 1993; Russo et al., 2005). Likewise, cells
that are tightly adhered to one another may prefer a purse string
based mechanism, while less adherent cells may drag themselves
forward over the wound matrix. In addition, large wounds in
tissue culture wound assays tend to heal by lamellipodia
extension and cell migration (Bement et al., 1993), whereas
smaller wounds heal by purse string contraction of an actomyosin
ring assembled at the wound edge (Bement et al., 1993). While
these different repair mechanisms have been associated with
specific contexts, a major challenge in the field is to identify the
molecular components underlying each repair mechanism and to
elucidate their roles in determining a particular tissue’s means of
Drosophila has recently emerged as a genetic model for
studying epithelium wound repair and the subsequent
inflammatory response (Wood et al., 2002; Galko and Krasnow,
2004; Mace et al., 2005; Stramer et al., 2005). Embryonic
(epithelial) wound repair has been proposed to take place using an
actin cable that operates as a purse string to draw the hole mostly
5984 Research Article
Journal of Cell Science
closed followed by the use of filopodia for the final knitting
together of the epithelium cells (Wood et al., 2002), whereas post-
embryonic (tissue) repair has been proposed to occur through cell
shape changes and cell migration (Galko and Krasnow, 2004;
Lesch et al., 2010). Furthermore, genetic screens for components
involved in embryonic (epithelial) and post-embryonic (tissue)
Fig. 1. See next page for legend.
Drosophila epithelial wound repair 5985
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wound repair have, with the notable exceptions of the JNK
signaling cascade and the Rho family of small GTPases, yielded
non-overlapping sets of mutants (Campos et al., 2010; Lesch et al.,
2010), reinforcing the idea that the regulatory mechanisms
employed in wound repair are context-dependent.
To determine the effect of different parameters on wound
repair mechanism, we employed 4D in vivo microscopy along
with mutant analysis to define the series of changes that occur
during four distinctive phases in response to epithelial wounding
in Drosophila embryos. We find that specific molecular
components including myosin, E-cadherin, Echinoid, the
plasma membrane, microtubules, and the Cdc42 small GTPase
respond dynamically during wound repair, and demonstrate that
perturbations of each of these components result in abnormal
wound healing. Our results show that embryo epithelial wound
repair requires a combination of partially redundant mechanisms
involving intertwined actin elements (dynamic cellular
protrusions and a contractile actomyosin cable), and provide
new insight into the different cellular machineries and
mechanisms required for these processes.
Epithelial wound repair occurs in four distinct phases in
the Drosophila embryo
Stage 15 Drosophila embryos have a single layer ectoderm
covering the surface of the embryo (Fig. 1B–F). Previous studies
have shown that while most of the embryo surface is subjected to
tensile forces due to dorsal closure movements, the ventral
epidermis is under lower net tension (Kiehart et al., 2000). Thus,
we generated wounds in the ventral epithelium by ablating a
circular patch of cells then followed the wound repair process by
4D confocal microscopy. The ablations were highly reproducible
(Fig. 1H), and XZ views showed the damage was restricted to the
epithelial cell layer, as the tissue underlying the epithelium
remains intact (Fig. 1G9). In vivo analysis of epithelial wound
repair in embryos expressing markers for actin (sGMCA;
spaghetti squash driven, GFP, moesin-a-helical-coiled and actin
binding site) and the nucleus (His2Av-mRFP), showed a range
of dynamic morphological changes in the epithelium cells
(Fig. 1G,H; Table 1). We found that the epithelial wound
repair process can be divided into four phases, corresponding
with the morphological changes observed: (i) expansion, (ii)
coalescence, (iii) contraction and (iv) closure.
Upon wounding (4–5 cells, ,800 mm
area), a clear gap is
visible in the epithelium (Fig. 1G,G9). During this initial
expansion phase, the tissue margins retract from the wound and
cells at the edge of the injured area exhibit different levels of
damage. Many cells at the edge of the wound showed high actin
levels, particularly at cell junctions, and became the leading edge
cells for the repair process (Fig. 1G; supplementary material
Movie 1A). Other cells, however, showed either lower actin
levels and fell below the plane of the epithelium, or became
rounded (swollen) and were removed from the wound edge
(Fig. 1G). These morphological changes suggest that some of the
epithelium cells suffer irreparable damage upon wounding, and
as a consequence need to be removed from the epithelium. We
examined if caspase activity, one of the hallmarks of cell death
by apoptosis, was activated in the cells at the wound border using
the caspase fluorescent sensor, Apoliner (Bardet et al., 2008).
Apoliner was expressed in the embryo epithelium, and its
localization was followed upon wounding (Fig. 1I). None of the
cells at the wound edge showed detectable translocation of GFP
into the nucleus, suggesting that caspases are not activated,
neither in the damaged cells nor the cells surrounding the wound
area. Thus, cell removal likely occurs by necrosis and/or cell
During the coalescence phase, the wound reaches its largest
size then begins to assemble the cellular machineries necessary
for closing the lesion. To be able to compare this phase
in different genetic backgrounds, we statistically defined
coalescence as the time in which the wound size remained
relatively constant at greater than 90% of the maximum area.
Actin foci were visible along the leading edge, particularly at
cells junctions, and, by 5 minutes post-wounding, dynamic
protrusions were detected on cells at the leading edge of the
wound (Fig. 1G,G9,L; supplementary material Movie 1A). Actin
accumulated initially in discontinuous patches at the apical edge
of these leading edge cells (Fig. 1J–K9; supplementary material
Movie 2A). During the next 10–15 minutes these discontinuous
actin patches joined up to form a thick and continuous actin cable
encircling the leading edge of the wound (Fig. 1G). As a result of
these events, the wound was observed to decrease slightly in size
and changed from a jagged irregularly shaped opening to a taut
rounded hole.
The contraction phase is characterized by the rapid reduction
in area of the rounded wound opening (Fig. 1G,G9;
supplementary material Movie 1A). For medium sized wounds
(,800 mm
) this process takes ,42 minutes on average
(Fig. 1H; Table 1). Actin enrichment in the cable increased
throughout this phase, indicative of the contractile force
associated with a functional actomyosin purse-string. Apically-
localized actin-rich protrusions, both filopodia and broad
lamellipodia, were observed throughout the contraction phase
in the leading edge cells. These protrusions actively probed the
open space of the wound area, making frequent contact with
Fig. 1. Phases of epithelial wound repair in Drosophila.(A) Schematic
diagram of multicellular wound repair. Damaged cells at the leading edge of
the wound will be either repaired (gray) or removed (white). Wound closure
can then proceed by lamellipodial crawling or contraction of an actin purse
string. (B–E)Drosophila embryo ectoderm is a single layer epithelium.
Confocal images of the lateral side (B), ventral side (C) and cross-section
(E) of stage 15 embryos expressing actin and nuclear markers (sGMCA and
His2Av-mRFP). In B and C anterior is left; in B dorsal is up. (D) Cross-
sections of a stage 15 embryo expressing GFP-a-tubulin in the epithelium
only. (F) Schematic diagram of a stage 15 embryo showing the organization
of the germ layers (adapted from Hartenstein, 1993). (G,G
) Time-lapse series
of surface projections (G) and cross-sections (G9) of a stage 15 embryo
expressing actin and nuclei markers (sGMCA and His2Av-mRFP). The
phases of wound repair are depicted. White arrows and arrowheads indicate
leading edge cells retracting and accumulating actin. Collapsing cells are
marked with asterisks. Filopodial extensions (blue arrows) and tethering (blue
arrowhead) are indicated. (G9) Yellow arrowheads mark the extent of wound
expansion. Hemocytes (yellow arrows) and the actin cable (green arrows) are
indicated. (H) Analysis of the wound healing response (n510; results are
given as means 6s.e.m.), showing the wound repair phases.
(I) Time-lapse series of an embryo expressing the caspase biosensor Apoliner.
GFP is not translocated to the nuclei of rounded cells (arrows). (J–L) Time-
lapse series of surface projections (J,K,L) and cross-sections (I9,J9) of a stage
15 embryo expressing actin (sGMCA). High magnification images (1006)
show filopodia probing the wound environment during the coalescence
(L) and contraction phases (K,K9; arrows). Scale bars: 20 mm
(C,G,I,J); 10 mm (B,D,E,G9,J9,K,K9); and 5 mm (L).
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apical protrusions from other leading edge cells, as well as
occasional contact with the overlying vitelline membrane
(Fig. 1K,K9). We did not observe basal protrusions or contact
of apical protrusions with the underlying somatic muscle cells
and/or ventral cord cells.
In the closure phase, the final ,5% of the peak wound area
was drawn closed to restore tissue integrity (Fig. 1G;
supplementary material Movie 1A). Similar to what has been
shown previously, we found that contacts between filopodia/
lamellipodia from opposing edges mediate this final wound
closure by knitting together the wound edges (Wood et al., 2002).
Wound size does not affect the mechanism of repair used
We observed both dynamic cellular protrusions and formation of
an actomyosin cable during the coalescence phase, suggesting
that Drosophila embryos may be able to repair lesions using
more than one mechanism. One of the factors proposed to affect
the mechanism by which wounds will be repaired is wound size.
To determine if this was a deciding factor in the Drosophila
embryo, we examined repair in different sized wounds (Fig. 2A–
I, Table 1). Wounds fell in three different size ranges (area
determined at fully expanded wound): (i) small: wound area
,500 mm
; (ii) medium: wound area ranging from 500-
1200 mm
; and (iii) large: wound area .1200 mm
. Much
larger sized wounds (.2500 mm
) can undergo successful
repair, but were not used here since they often trigger an
accompanying stress response complicating subsequent analyses.
We found that in wild-type embryos, wound repair kinetics
followed the same trends (see percentage of time in each phase)
and different sized wounds displayed similar phenotypic repair
dynamics (i.e. using an actin cable and actin-rich protrusions)
(Fig. 2A–I; Table 1; and data not shown).
Table 1. Wound size does not affect epithelial wound repair dynamics in wild-type embryos
Size Total
Expansion Coalescence Contraction
) Time % Time % Time % Time %
Wild type
,500 32.761.7 100 2.260.2 6.760.5 3.860.3 12.061.4 27.261.7 82.861.4
Wild type 500–1200 50.463.8 100 3.160.5 5.960.7 5.060.5 10.261.2 42.363.3 83.961.1
Wild type .1200 103.3613.4 100 5.761.7 5.461.6 9.762.4 9.662.4 88.0612.9 84.963.8
Wild-type embryos express sGMCA.
Total time is determined as the time in expansion, coalescence and contraction. Closure is excluded because the precision of measuring openings of ,15 um
is unreliable.
Fig. 2. Parameters of epithelial wound repair. (A–I) Parameters of the epithelial wound response (all results are given as means 6s.e.m.). (A) Effects of
wound size on wound repair dynamics (small, n55; medium, n510; large, n55). Analysis of each phase of repair: expansion and coalescence by area (B,D,F) and
duration (C,E,G); contraction phase by area (H) and duration (I). (J–L) Effects of embryo mounting techniques on wound repair. Single slice micrograph at the
plane of the wound in membrane (J) and coverslip (K) mounted embryos expressing actin (sGMCA). (L) Analysis of wound repair dynamics using membrane
(blue) and coverslip (red) mounting. Scale bar: 20 mm.
Drosophila epithelial wound repair 5987
Journal of Cell Science
We were surprised, however, to find that the method of
mounting the embryos for wounding and imaging greatly affected
the outcome of the wound repair dynamics (Fig. 2J–L). To
perform laser wound assays, embryos were initially mounted
either: (i) in series 700 halocarbon oil between a hydrophilic gas
permeable membrane and a coverslip (a popularly used mounting
method; Wood and Jacinto, 2005), or (ii) on a glue-painted
coverslip then covered with halocarbon oil (Foe et al., 2000). The
epithelial wound response was highly reproducible using glue
mounting (Fig. 2L). However, embryos mounted using the
membrane exhibited a highly variable repair response and in
some cases even failed to heal (Fig. 2L). This interference with
wound repair is likely caused by the slight compression
introduced when embryos are sandwiched between the
membrane and coverslip (Fig. 2J,K). Thus, our wound assays
were typically conducted using medium size wounds with
embryos mounted on glue painted coverslips. Under these
conditions, wound repair was completed on average by
50 minutes (Table 1).
Myosin is required for the contraction of the actomyosin
purse string
Actomyosin-powered purse string contraction is a mechanism
common to multiple morphogenetic movements in a variety of
organisms (Young et al., 1993; Williams-Masson et al., 1997;
Vavylonis et al., 2008), and has been suggested to power wound
closure in Drosophila embryonic epithelium wounds (Wood et al.,
2002). To further understand the role of the actomyosin cable, we
examined the function of myosin II in vivo and its contribution to
the wound repair process. We first monitored the dynamics of
myosin II recruitment relative to actin by examining embryos co-
expressing a myosin regulatory light chain (sqh) fusion to GFP
(sqh-GFP) and actin (sChMCA). Myosin II can be detected at the
wound edge 5 minutes after wounding, overlapping with actin
accumulation along the leading edge (Fig. 3A–B9; supplementary
material Movie 1B). During contraction, actin and myosin co-
localized to form a robust actomyosin purse-string in both surface
and orthogonal planes. A striking feature of this accumulation
was the complete absence of myosin II from the actin-rich
protrusions (Fig. 3A–B9), suggesting that myosin II is only
associating with actin as part of the supracellular cable.
Given myosin dynamics in response to wounding, we
anticipated that mutations affecting myosin II would impair
actomyosin purse string assembly and contractility without
affecting the actin-rich protrusions. In zipper
homozygous mutants, which disrupt the myosin II heavy chain
gene, an actomyosin purse string is not assembled at the leading
edge of the wound (Fig. 3C,C9; Table 2; supplementary material
Movie 2B). The wound edges were irregular, with actin
accumulated in patches that extended over a few cells and
particularly at cell-cell junctions, but never forming the thick
continuous cable that encircles wounds in wild-type embryos
(Fig. 3C,C9). Interestingly, despite the lack of a contiguous actin
cable, wounds in zip
mutant embryos healed, albeit with a severe
delay especially in the contraction phase (P50.0051; Fig. 3D,E;
Table 2). In vivo analysis showed that wound closure was
achieved by the actin-rich cellular protrusions (Fig. 3C,C9,F;
supplementary material Movie 2B). Quantification of the wound
area covered by protrusions showed that zip
embryos displayed
three times more protrusions than wild-type embryos at similar
stages of repair (Fig. 3G). This increase could be detected from
the coalescence phase (100% of max wound area) (Fig. 3F,G).
These protrusions pulled the wound closed by capturing
protrusions from adjacent or opposing cells and tugging these
areas closer together. As a consequence, reduction of the wound
area occurred in jumps (Fig. 3D; supplementary material Fig.
S1C). Our data suggests that the contractile force of the
actomyosin purse string was necessary for the rapid closure of
the wound during the contraction phase, but in the absence of this
component, both local and long-distance zippering by dynamic
filopodia and lamellipodia can mediate wound closure.
Cadherin-based adherens junctions are necessary to
mediate the actomyosin purse string contraction
In order for the actomyosin purse string to contract to close
a wound it needs to be linked from cell to cell to generate a
supracellular cable. Cadherin-based adherens junctions are a
major site of cell-cell adhesion and actomyosin network
anchoring to the plasma membrane within a cell (reviewed in
Tepass et al., 2001; Hartsock and Nelson, 2008). Analysis of
epithelium wound repair in embryos expressing markers for E-
Cadherin (DE-Cadherin-GFP) and actin (sGMCA) showed that
E-Cadherin accumulated at the apical most side of the leading
edge cells and in particular at the sites of lateral cell-cell
junctions, consistent with its reported role (Fig. 4A,A09;
supplementary material Movie 1C) (Wood et al., 2002). By
midway through the contraction phase, cadherin expression was
largely lost along the apical most side of leading edge cells,
remaining at the sites of lateral cell-cell junctions (Fig. 4B,B9).
Interestingly, we found that immediately following injury, some
of the cells at the wound site retained DE-Cadherin expression
but lacked actin (Fig. 4A). These cells slowly disappeared from
the surface of the embryo as the leading edge cells moved
forward to close the wound (Fig. 4A,A9).
Given the accumulation of DE-Cadherin at the sites of cell-cell
junctions and its overlap with actin accumulation, perturbations
in the levels of cadherin would be expected to disrupt cable
function and possibly its assembly. Cadherin loss of function
mutants (shotgun;shg
) showed defects in wound repair
during the contraction phase: actin cable assembly was impaired
leading to initially jagged wound boundaries and irregularly
shaped wounds (Fig. 4C,C9,E,F; supplementary material Fig.
S1A; Table 2). Remarkably, wound closure was eventually
achieved by non-uniform contraction of leading edge areas (a
few cells wide) that accumulated actin. These areas of localized
pinching often corresponded with cells exhibiting active
protrusions (Fig. 4D; supplementary material Movie 2C). Our
data shows that DE-Cadherin is required for both the assembly
and stability of the actin purse-string at the wound leading edge.
Echinoid is required for actomyosin purse string assembly
and stabilization
Another adherens junction-associated cell adhesion molecule, the
Drosophila nectin ortholog Echinoid (Ed), has been shown to
interact with Canoe (afadin) and Jaguar (unconventional myosin
VI) to modulate the accumulation of actin regulators at the
leading edge during dorsal closure (Wei et al., 2005; Laplante
and Nilson, 2006; Lin et al., 2007; Chang et al., 2011; Laplante
and Nilson, 2011). In this context, Ed has been proposed to
mediate cell sorting by promoting actomyosin cable formation
and filopodial protrusions in the dorsal most leading edge cells.
In the context of epithelial wound repair, we anticipated that loss
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Fig. 3. Myosin II is required for assembly of an actin purse string. (A–B
) Time-lapse series of surface projections (A,A9) and cross-sections (A0) of a stage 15
embryo expressing myosin and actin markers (sqh-GFP and sChMCA). Myosin accumulates at the leading edge of epithelium wounds overlapping with the actin
cable. (B,B9) High magnification views (white squares in A) showing myosin and actin overlapping at the leading edge. Myosin II is absent in the
protrusions (arrows). (C,C
) Time-lapse series of stage 15 zip
mutant embryo expressing an actin marker (sGMCA), and showing incomplete actin cable
assembly. Surface projections (C) and cross-sections (C9) are shown. (D,E) Quantification of wound area over time (wild type, n510; zip
,n54; results are given
as means 6s.e.m.) for all phases of wound repair (D) and an expansion of the first 20 minutes (E). (F) High magnification (1006) views of protrusions in wild-
type and zip
wounds at time-points representing 100, 75 and 50% of the maximum wound area. (G) Quantification of the area of protrusions (wild type, n54;
,n54), P-values are indicated. Scale bars: 20 mm (A,A9,C) and 10 mm(A0,C9,F).
Table 2. Comparison of medium-sized wound repair in wild-type and mutant embryos
Allele Total time
Expansion Coalescence Contraction
Time P-value Time P-value Time P-value
Wild type
50.463.8 – 3.160.5 – 5.060.5 – 42.363.3 –
164.8614.8 0.005 4.561.3 0.25 7.561.7 0.08 152.7614.5 0.0051
106.8611.6 0.0037 5.261.2 0.09 5.761.2 0.57 96.0610.3 0.0025
105.266.5 ,0.0001 4.061.1 0.26 13.061.2 ,0.0001 88.266.8 0.0005
55.264.0 0.4787 3.760.9 0.57 10.361.9 0.045 41.562.7 0.87
107612.9 0.0140 2.860.6 0.73 8.462.0 0.17 95.8611.4 ,0.0001
All embryos express sGMCA.
Total time is determined as the time in expansion, coalescence and contraction.
Significant if P,0.05 (bold).
Drosophila epithelial wound repair 5989
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of Ed might prevent epithelial wound repair by destabilizing
adherens junction complexes, disrupting actomyosin purse string
assembly and stabilization, and by reducing cellular protrusions
from leading edge cells. Consistent with this, ed
embryos failed to form a continuous actomyosin purse-string at
the leading edge of wounds (Fig. 4G,G9,K–L9), and exhibited
significant delays in both the coalescence and contraction phases
(P,0.0001 and P50.0005, respectively) (Fig. 4I,J; Table 2;
supplementary material Fig. S1A and Movie 2D). However,
similar to cadherin mutants, wound closure took place by local
contraction of actin patches and protrusions. Indeed, we observed
protrusions that joined with those from nearby and/or opposing
cells to pinch the wound shut (Fig. 4H). Thus, Ed in the context
of wound repair may be required for the stabilization of adherens
junctions needed to link the actomyosin cable intercellularly at
the wound leading edge cells.
Actin-rich protrusions are present throughout the
epithelial repair process
Previous studies suggested that an actomyosin cable was needed
to close the edges of the wound until the fronts were close enough
for cellular protrusions to reach across and knit closed the small
remaining hole (Wood et al., 2002). In contrast to previous
reports, we observed filopodia and lamellipodia throughout the
wound repair process in wild-type embryos making contact with
other protrusions (Fig. 1I–J9; Fig. 4B9). To determine the role of
cellular protrusions during the contraction phase when the
actomyosin cable is thought to be the major mechanism for
closing the hole, we followed the dynamics of cellular
protrusions during the wound repair process. In particular, we
examined two membrane markers: GFP-PH-PLC, the pleckstrin-
homology domain of PLC fused to GFP, which specifically binds
to the phosphoinositidine PI(4,5)P2 and is enriched in the apical
side of epithelial cells (Pinal et al., 2006), and Venus-GAP43, the
palmitoylation signal of the growth-associated protein 43 fused to
Venus fluorescent protein, which tightly anchors to the plasma
membrane (Mavrakis et al., 2009). In addition, we examined a
marker for microtubules as they have previously been reported to
be reorganized in response to injury (Etienne-Manneville, 2004).
Cells at the leading edge of the wounds show highly dynamic
filopodia and lamellipodia enriched in both membrane markers,
particularly when the PH-PLC reporter was used (Fig. 5A,B).
These cellular extensions reach across the wound to make both
nearby and long-distance connections to the protrusions of other
cells (Fig. 5A,B; supplementary material Movie 3). In addition,
GAP43 was observed to accumulate in the cell-cell contacts
(Fig. 5C–C0).
Time-lapse movie analysis of Drosophila embryos expressing
GFP-a-tubulin in the epithelium shows that, unlike actin,
microtubules do not accumulate at the leading edge of the
wound (Fig. 5D–F). However, consistent with previous
observations during the dorsal closure process (Jankovics and
Brunner, 2006; Liu et al., 2008), microtubules are recruited to the
filopodial protrusions assembled by the leading edge cells
(Fig. 5D–F). We wounded embryos mutant for the microtubule
capping protein EB1 (eb1
), which disrupts microtubule
dynamics (Elliott et al., 2005). Interestingly, we observed
defects only in the coalescence phase, where eb1
embryos showed a significant delay before beginning contraction
(P50.045; Fig. 5G–I; Table 2). We are not able to rule out,
however, that disruption of microtubule dynamics in this context
is indirect and may not affect cellular protrusions per se, but
rather affect repair during the coalescence phase by impairing
the recruitment of the repair machineries/components thereby
delaying initiation of the contraction phase.
Actin-rich protrusions are required for embryo epithelial
wound closure
To further investigate the role of dynamic cellular protrusions, we
examined the function of the Cdc42 small GTPase in vivo and its
contribution to the wound repair process. Cdc42 constitutive
active or dominant negative mutants have been shown to regulate
filopodial assembly in tissue culture cells and in both dorsal
closure and wound repair in Drosophila (Nobes and Hall, 1995;
Jacinto et al., 2000; Wood et al., 2002). We first generated a
ChFP-Cdc42 reporter to follow Cdc42 localization in vivo
simultaneously with actin (ChFP-Cdc42; SGMCA) (Fig. 6A–
A0). Throughout wound repair, Cdc42 did not accumulate at the
wound leading edge or in the actin-rich protrusions, suggesting
that upon wounding Cdc42 subcellular localization does not
change significantly.
To modulate the levels of Cdc42, we used the heteroallelic
combination Cdc42
. These Cdc42 loss of function
mutant embryos exhibit severe developmental defects as
previously reported (Genova et al., 2000), where epithelial cells
fail to elongate into columnar shaped cells, resulting in failure of
germ band retraction and, in most cases, random holes appeared
in the epithelium. Upon wounding, Cdc42
embryos were
not significantly delayed in the expansion or coalescence phases
(Fig. 6B–D; Table 2). By contraction, these wounds became
quite rounded consistent with the presence of circumferential
tension provided by a functional actomyosin cable (Fig. 6B,B9;
supplementary material Fig. S1B and Movie 2E). Strikingly, we
observed a severe reduction of filopodia and lamellipodia
protrusions in the leading edge cells throughout the wound
repair process: Cdc42
mutant embryos displayed three
times less filopodia/lamellipodia than wild-type controls
(Fig. 6E,F). As a consequence, these embryos spend
95 minutes in the contraction phase, over double the time spent
by wild-type embryos (Fig. 6D; Table 2). As previously
described, Cdc42
mutant embryos are unable to seal
the wound during the final closure phase and a small hole persists
(n54/5 embryos) (Fig. 6B) (Wood et al., 2002). Our data shows
that the contractile force of the actomyosin cable was sufficient to
draw the wound closed, however the kinetics of this phase were
not the same as those observed for repair in wild-type embryos,
indicating that cellular protrusions are playing an active role in
the contraction phase.
Epithelial wound repair requires both assembly of an actin
purse string and actin-rich protrusions
Our in vivo mutant analysis allowed us to dissect the individual
contributions of cytoskeleton components to the wound repair
process. In particular, our results show that disruption of the
actomyosin cable leads to increased protrusions (Fig. 3C–G),
whereas disruption of cellular protrusions leads to defects in both
active wound closure during contraction and the final wound
zippering (Fig. 6B–E). To determine if both components the
actomyosin cable and cellular protrusions – are required
simultaneously for epithelial wound repair, we generated
double mutants that disrupts both structures by targeting
myosin II and Cdc42 small GTPase. Since generating double
Journal of Cell Science 125 (24)5990
Journal of Cell Science
genetic mutants was not possible, we used the combination of a
genetic mutant, zip
and dsRNA interference (RNAi) to
specifically reduce Cdc42 (Cdc42
). We injected dsRNA for
Cdc42 into zip
mutant and wild-type embryos, or buffer alone
into zip
mutants (see Material and Methods). Cdc42
embryos display similar wound repair dynamics when
Fig. 4. See next page for legend.
Drosophila epithelial wound repair 5991
Journal of Cell Science
compared to Cdc42
mutants (supplementary material
Fig. S1D), and buffer-injected zip
homozygous mutant embryos
display similar wound repair dynamics to uninjected zip
homozygous mutants (supplementary material Fig. S1E). zip
double mutants fail to close epithelium wounds:
100% of the wounds are still open at 220 minutes post-wounding,
the time by which zip
and Cdc42
single mutants are
closed (Fig. 6G,H). zip
double mutant embryos show
defects in all the wound repair phases, particularly in the
coalescence and contraction phases (Fig. 6G,H). During the
coalescence phase, wound edges were jagged and wound shape
was irregular, with actin failing to accumulate in the leading edge
cells. Strikingly, patches of continuous actin are not observed
until 60 minutes post-wounding (four times longer than wild-type
embryos) (Fig. 6G). The contraction phase is severely delayed,
due to the lack of both a proper actin cable and cellular
protrusions (Fig. 6G). Significantly, zip
mutant embryos stall during the contraction phase, and a hole
persists with an average area of 159.1631.5 mm
(n54). Thus,
our data shows that a combination of functional actomyosin cable
and cellular protrusions are required for proper embryo epithelial
wound repair.
Epithelial wound repair in the Drosophila embryo proceeds
through a distinct set of phases and requires the coordinated
efforts of several cytoskeletal components/machineries. We
show, for the first time, the specific contributions of myosin, E-
cadherin, Echinoid, and plasma membrane components in
response to wounding (Fig. 7A–C). Actin-rich protrusions are
the first cytoskeleton structure observed early on in the wound
repair process, followed by the formation of an actomyosin
purse-string at the wound leading edge (Fig. 7A–C). While
each of these components by themselves makes distinct
functional contributions, they function together in the wild-
type wound repair process to ensure rapid and complete
Wound repair occurs in distinct phases
A major observation while studying embryo epithelial wound
healing in vivo is the existence of defined phases occurring at
characteristic intervals along the wound repair process, and
requiring specific molecular components. The expansion phase
encompasses the initial clearance of the wound area and the
establishment of the wound leading edge. Interestingly, none of
the mutants we examined significantly affected this phase,
suggesting that the factors affecting expansion may be inherent to
the tissue. Notably, the expansion phase was not affected in zip
mutant embryos, despite the epithelial cells in this mutant being
smaller on their apical surfaces. In addition, in severe E-cadherin
) mutant embryos in which only non-continuous patches of
epithelium exist by later stages of development, wounds expand
with normal dynamics, precluding morphogenetic tension as the
explanation for the initial wound expansion (data not shown). We
anticipate that mutants for components of the mechanosensory
system and/or signals that initiate the wound repair response will
impair the expansion phase.
We initially hypothesized that the coalescence phase
corresponded to a passive transition where the forces exerted
by the expanding wound were gradually overcome by the
contraction machinery. However, the delay we observed in eb1
mutants is consistent with this phase being active. Furthermore,
the coalescence phase is not extended in all mutants affecting the
actomyosin cable, as would be expected if the contractile forces
needed to overcome expansion are impaired.
The majority of wound closure is achieved during the
contraction phase, as the wound is actively closed by a
combination of cable contraction and cellular protrusions. The
wound area exhibits exponential rather than linear reduction,
consistent with a steady rate of wound margin ingression towards
the center. Interestingly, in mutants lacking the actomyosin
cable, the wound area is reduced in a more stepwise fashion
corresponding to local zippering events. This suggests that the
circumferential tension of the actomyosin cable not only provides
direct force for repair, but also transmits the force generated by
local zippering around the wound edge.
The final stage of repair is closure, in which filopodia and
lamellipodia on opposing epithelial faces contact and engage
to fuse the wound fronts and restore the continuity of
the epithelium. Consistent with previous reports, our genetic
analysis shows that the Cdc42 small GTPase is crucial in this
phase of the process, by modulating the formation of polarized
actin-rich protrusions (Wood et al., 2002). It will be interesting to
see if trafficking of adhesion molecules, or perhaps integrins,
along with filopodia are necessary for the protrusions to mediate
epithelial fusion and the formation of new cell-cell junctions.
Dynamic cellular protrusions play a role throughout the
wound repair process
In the absence of a functioning actomyosin cable, epithelium
wounds heal by extension and zippering of actin-rich cellular
protrusions. Previous studies have suggested that interactions
between filopodia and lamellipodia of neighboring cells were
only responsible for wound closure during the final steps of
repair, when the edges of the wound were close to one another
(Wood et al., 2002). In their study, wounding of Rho1 small
GTPase embryos, in which the actomyosin cable is disrupted,
showed a two-hour delay in repair during which no changes were
observed in the leading edge cells, followed by the wound closing
Fig. 4. Intact adherens junctions are required to anchor the actomyosin
cable. (A–B
) Time-lapse series of surface projections (A–A0,B,B9) and cross-
sections (A90) in wild-type embryos expressing DE-cadherin-GFP and actin
(sChMCA). Cells retaining E-cadherin but lacking actin are indicated (white
arrowheads). (B,B9) High magnification views (white squares in A) showing
actin and E-cadherin overlaps at the leading edge junctions. (C–D) Wound
repair in a shg
mutant embryo expressing an actin marker (sGMCA).
Time-lapse series of surface projections (C,D) and cross-sections (C9). High
magnification views of shg
embryos showing repair by localized
protrusion zippering between neighbor leading edge cells (D, white brackets).
(E,F) Quantification of wound area over time (wild type, n510; shg
n56; results are given as means 6s.e.m.) for all phases of wound repair
(E) and an expansion of the first 20 minutes (F). (G–H) Time-lapse series of
surface projections (G,H) and cross-sections (G9)ofaned
mutant embryo
expressing an actin marker (sGMCA). ed
protrusions contact one another,
resulting in local contractions of the wound edge (H, arrows).
(I,J) Quantification of wound area over time (wild type, n510; ed
,n55) for
all phases of wound repair (I) and an expansion of the first 20 minutes (J).
mutants assemble partial actomyosin cables at the wound leading
edge. Myosin II (a-non muscle myosin, MHC) accumulates at the wound edge
of wild-type (K,K9) and ed
mutants (L,L9) overlapping with actin
(phalloidin). Scale bars: 20 mm (A–A0,C,G); 10 mm(A09 ,C9,D,H,G9); and
5mm (K–L9).
Journal of Cell Science 125 (24)5992
Journal of Cell Science
with normal wound kinetics mediated by local zippering (Wood
et al., 2002). In contrast, our in vivo studies show that actin-rich
protrusions are present throughout the wound repair process,
from the onset of the coalescence through the closure phase.
These apically-localized protrusions are highly dynamic, as
observed with actin and plasma membrane markers, and reach
across the wound fronts throughout the repair process to contact
neighboring cells, as well as the overlying vitelline membrane.
When we disrupted the actomyosin cable using myosin (zip) and
E-cadherin (shg) mutants, we find that wound repair is
significantly delayed, and showed that actin-rich protrusions are
used for contraction in these mutants. Indeed, we observed long-
range events where filopodia reaching far across the middle of
the wound made contact and pulled the wound closed at the
middle. Consistent with this, we observe a delay in contraction
when the ability to form cellular protrusions, but not a functional
actomyosin cable, was impaired using Cdc42 mutants (Table 2).
The results observed with the Cdc42 mutants highlight two
important aspects of the mechanisms involved in the repair
process. First, protrusions are necessary for normal wound repair
kinetics during contraction. Second, intact protrusions are crucial
for the closure phase. In the absence of protrusions, the wound
fronts fail to fuse indicating that the contractile force of the
actomyosin cable cannot compensate for the lack of protrusions
in the final steps of wound closure.
Drosophila epithelial wound repair incorporates both actin
purse string and lamellipodial crawling mechanisms
Wound repair by lamellipodial crawling was thought to be an
exclusive repair mechanism of adult tissues, whereas embryonic
epithelium wound closure was mediated solely by actomyosin
purse string contraction (reviewed in Jacinto et al., 2000).
However, more recent studies have shown an overlap between
these two types of epithelial events and have suggested that
wound size and intrinsic tissue dynamics may also influence the
mode of wound closure (Bement et al., 1993; Wood et al., 2002;
Russo et al., 2005). We find that the repair of epithelial wounds
in the Drosophila embryo integrates both repair mechanisms. By
Fig. 5. Dynamic protrusions are assembled at the leading edge of epithelial wounds. (A–C
) Time-lapse series of surface projections (A,A0,B,C,C0) and
cross-sections (A9,C9) of embryos expressing the membrane reporters PH-PLC-GFP (A–B) or Venus-GAP43 (C–C0). High magnification view of protrusions in
embryos expressing PH-PLC-GFP (A0,B) and Venus-GAP43 (C0). Membrane in protrusions (arrows) and accumulation at cell-cell junctions (arrowheads) are
indicated. (D–F) Time-lapse series of embryos expressing GFP-a-tubulin in the epithelium, showing microtubules in cell protrusions (arrows). High magnification
views in E,F. (G,G
) Time-lapse series of surface projections (G) and cross-sections (G9) of a stage 15 eb1
embryo expressing an actin marker (sGMCA) showing
delayed actin cable assembly. (H,I) Quantification of the wound area over time (wild type, n510; eb1
,n56; results are given as means 6s.e.m.) for all phases
of wound repair (H) and an expansion of the first 20 minutes (I). Scale bars: 20 mm (A,C,D,G) and 10 mm(A9,A0,B,C9,C0,E,F,G9).
Drosophila epithelial wound repair 5993
Journal of Cell Science
5 minutes post-wounding the major structures observed at the
apical side of the leading edge cells are small protrusions, not the
actomyosin purse string. During the contraction phase, dynamic
filopodia extending all around the wound edge area can be seen
along with the prominent actomyosin cable. Impairing the
actomyosin cable leads to healing through cellular protrusions.
Fig. 6. Cdc42 is required for normal protrusion activity and, together with Myosin II, for proper epithelial repair. (A–A
) Confocal time series following
wound repair in stage 15 embryos coexpressing ChFP-Cdc42 (A9) and actin (sGMCA). (B,B
) Time-lapse series of wound repair in Cdc42
embryos expressing an actin marker (sGMCA). Notice the lack of protrusions at the leading edge cells. (C,D) Quantification of the wound area over time (wild
type, n510; Cdc42
,n55; results are given as means 6s.e.m.) for all phases of wound repair (C) and an expansion of the first 20 minutes (D). (E) High
magnification views (1006) showing the protrusions at the leading edge cells in wild-type and Cdc42
embryos at 100, 75 and 50% maximum wound area.
(F) Quantification of the area of protrusions, P-values are indicated (wild type, n54; Cdc42
,n54). (G,G
) Time-lapse series of wound repair in zip
double mutant embryos followed with an actin marker (sGMCA). Actin cable assembly, as well as cellular protrusions, are severely disrupted.
(H) Quantification of the wound area over time (wild type, n510; zip
,n54; Cdc42
,n55, zip
,n54; results are given as means 6s.e.m.).
Scale bars: 20 mm (A,A9,B,G) and 10 mm(A0,B9,E,G9).
Journal of Cell Science 125 (24)5994
Journal of Cell Science
Likewise, impairing cellular protrusions leads to healing through
actomyosin cable contraction. Interestingly, shg mutant embryos,
which have an impaired actomyosin cable, and Cdc42 mutants,
which have reduced actin-rich cellular protrusions, spend almost
identical amounts of time in the contraction phase suggesting that
the two mechanisms are equally important to generate the
contractile force required for wound closure. In both cases, the
repair kinetics are severely delayed compared to wild type, again
suggesting that both mechanisms are working synergistically in
wild-type repair. Consistent with this simultaneous requirement
for both an actin cable and cellular protrusions for proper repair,
embryos in which both wound repair mechanisms are
functionally disrupted (by combining mutants for Myosin II
and Cdc42) fail to close epithelium wounds.
While our knowledge of the dynamics and components of
epithelial wound repair has rapidly increased in recent years with the
addition of new model organisms and imaging techniques, major
questions still remain concerning the intrinsic factors modulating the
repair response, as well as the signaling molecules involved in
triggering and terminating wound repair. In particular, what are the
Fig. 7. Working model for epithelial wound
repair in the Drosophila embryo.
(A,B) Schematic of surface projection (A) and
cross-section (B) views depicting the dynamic
behavior of embryo epithelium cells in
response to wounds, as well as the molecular
components recruited during the wound repair
process. Shape changes and behavior for
different cells (indicated by letters a–e) at the
wound edge are indicated. (C) Phases and
components of the epithelial wound response.
Drosophila epithelial wound repair 5995
Journal of Cell Science
signals that lead to the context-dependent choice of wound repair
mechanism? The Drosophila embryo is an excellent genetic model
in which these questions encompassing the complex process of
epithelial wound repair can be systematically addressed.
Materials and Methods
Fly strains and genetics
Flies were cultured and crossed at 25˚
C on yeast-cornmeal-molasses-malt medium.
The following stocks containing fluorescence fusion proteins were used: sGMCA
(Kiehart et al., 2000), sChMCA (Abreu-Blanco et al., 2011), P{His2Av-
mRFP1}III.1 and P{UASp-GFPS65C-alphaTub84B} (Bloomington Stock
Center), P{UAS-Apoliner}8/TM3, Sb (Bardet et al., 2008), sqh
; P{sqh-
GFP}42 (Royou et al., 2004), ubi-DE-Cadherin-GFP (Oda and Tsukita, 2001,
Kyoto Stock Center), P{UASt-GPF-PH-PLC} (Pinal et al., 2006), UASp-Venus-
GAP43 (Mavrakis et al., 2009), and ChFP-Cdc42 (this study). Double
fluorescently tagged lines were generated using standard genetic methods.
Expression of UAS lines was driven in the embryo epithelium with P{en2.4-
GAL4}e22c (Bloomington Stock Center).
The following mutants alleles were used: Cdc42
and Cdc42
(Fehon et al.,
1997), ed
(Laplante and Nilson, 2006), and from the Bloomington Stock Center:
(Young et al., 1993), shg
and shg
(Tepass et al., 1996), and eb1
et al., 2005). Mutant alleles were crossed to the sGMCA; CyO-ChFP or sGMCA;
TM3-ChFP balancer stocks, to screen for homozygous mutants by selecting
against the ChFP balancer. ed
mutants are germ-line clones, generated using the
FLP-DFS system with ed
FRT40A (Laplante and Nilson, 2006). Mutant
embryos for Cdc42 were generated using the heteroallelic combination Cdc42
(Fehon et al., 1997). All mutant embryos express sGMCA allowing the
actin cytoskeleton to be followed.
Generation of ChFP-Cdc42 and CyoChFP constructs and transgenics
ChFP-Cdc42 is a fusion of ChFP with the Cdc42 cDNA, expressed under the control
of the spaghetti squash (sqh) promoter. The construct was generated as follows: the
Cdc42 ORF was amplified by PCR from DGCr1 cDNA HL08128, then cloned 39of
ChFP as a EcoRI and XbaI fragment. The ChFP-Cdc42 fusion was PCR amplified
and cloned into the StuI and XbaI sites of the pSqh59+39UTR plasmid (Abreu-Blanco
et al., 2011). To generate CyO-ChFP and TM3-ChFP balancers, ChFP was cloned
into the StuI and XbaI sites of the pSqh59+39UTR plasmid (Abreu-Blanco et al.,
2011). These constructs were used to make germline transformants as previously
described (Spradling, 1986). The resulting transgenic lines were mapped to a single
chromosome and shown to have non-lethal insertions.
RNA interference (RNAi) assays
To generate the Cdc42 double strand RNA (dsRNA), the template was amplified
by PCR using primers that include the T7 promoter sequence. The sequence used
for the Cdc42 dsRNA was selected based on previously reported RNAi lines by the
TRiP stock center (DRSC25134). The dsRNA was synthesized and injected into
embryos as previously described, at a final concentration of 2.5 mM (Magie et al.,
2002; Magie and Parkhurst, 2005). Embryos were injected and aged at room
temperature for 12–14 hours before conducting the wound healing assays. Control
(sGMCA) and zip
(sGMCA, ChFP balancer) were injected with the Cdc42
dsRNA, generating Cdc42
and zip
embryos, respectively. Control
(buffered injected) zip
was also included in the assay.
Stage 15 laser wounded embryos were recovered, fixed with 37% paraformaldehyde/
heptane for 5 minutes, and hand devitellinized. Immunofluorescence was performed
as described previously (Abreu-Blanco et al., 2011) with anti-nonmuscle myosin
antibody (1:500; Young et al., 1993) and anti-rabbit Alexa Fluor 568 (1:1000;
Invitrogen). Alexa-Fluor-488-labeled phalloidin (Invitrogen) was used at 5 Units/
assay and added with the secondary antisera. Samples were mounted in SlowFade
Gold (Invitrogen/Molecular Probes).
Confocal fluorescent microscopy
Confocal microscopy was performed using a Zeiss LSM-510M microscope (Carl
Zeiss Inc., Jena, Germany) with excitation at 488 nm or 543 nm, and emission
collection with BP-500-550 or LP560 filters, respectively. A Plan-Apochromat
2060.75 dry objective was used for imaging. Images were processed in ImageJ
(http://rsb.info.nih.gov/ij/), and assembled with Canvas 8 software (Deneva
Systems, Inc.).
Live imaging
All imaging was performed at room temperature (23˚
C). Stage 15 embryos were
hand dechorionated, dried for 5 minutes then transferred individually with forceps
onto strips of glue dried onto No. 1.5 coverslips, and covered with series 700
halocarbon oil (Halocarbon Products Corp.) (Foe et al., 2000). The following
microscopes were used: (i) Nikon TE2000-E stand (Nikon Instruments, Melville,
NY), with 4061.4 NA objective lens, controlled by Volocity software (v.5.3.0,
PerkinElmer, Waltham, MA). Images were acquired with 491 nm and 561 nm
lasers, with a Yokogawa CSU-10 confocal spinning disc head equipped with a 1.56
magnifying lens, and a Hamamatsu C9100-13 EMCCD camera (PerkinElmer,
Waltham, MA). (ii) UltraVIEW VoX Confocal Imaging System (PerkinElmer,
Waltham, MA), in a Nikon Eclipse Ti stand (Nikon Instruments, Melville, NY), with
6061.4 NA or 10061.4 NA objective lens and controlled by Volocity software
(v.5.3.0, PerkinElmer, Waltham, MA). Images were acquired with 491 nm and
561 nm, with a Yokogawa CSU-X1 confocal spinning disc head equipped with a
Hamamatsu C9100-13 EMCCD camera (PerkinElmer, Waltham, MA). (iii) Nikon
LiveScan Swept Field Confocal (for Nikon by Prairie Technologies Inc., Middleton,
WI) mounted on a Nikon Eclipse Ti (Nikon Instruments, Melville, NY); with 606
1.4 NA objectives lens, using the NIS-Elements AR 3.0 as acquisition software
(Nikon Instruments, Melville, NY). Images were acquired with a 491 nm laser, and a
Photometrics QuantEM: 512SC EMCCD camera (Photometrics, Tucson, AZ). All
images acquired with a 406or 606objective lens are 25 mm stacks/0.5 mm steps, for
the 1006images the stacks correspond to 1.5 mm/0.25 mm steps.
Laser wounding
Laser ablation experiments used the Photonic Instruments MicropointHComputer
Controlled system (Photonic Instruments, St Charles, IL), as previously described
(Abreu-Blanco et al., 2011).
Image processing, analysis and quantification
Image series were either analyzed with Volocity software (v.5.3.0, PerkinElmer,
Waltham, MA), or were exported as TIFF files then imported into ImageJ for
processing. XY projections of 1–5 mm were generated. Wound areas were
measured manually with ImageJ or NIS-Elements AR software (version 3.0, Nikon
Instruments, Melville, NY). Protrusion area was measured manually at 100, 75 and
50% of the maximum wound area with ImageJ. Circularity was measured using the
following formula: 4p6area/perimeter
, in which a perfect circle has a value of 1.
A Student’s ttest was used to analyze the data; P,0.05 was considered to be
statistically significant. All graphs present values 6s.e.m. All measurements were
downloaded into Microsoft Excel and the data were graphed using Prism 5.0c
(GraphPad Software).
We thank Parkhurst laboratory members for their interest, advice and
comments on the manuscript. We are very grateful to D. Brunner, R.
Fehon, R.E. Karess, D.P. Kiehart, J. Lippincott-Schwartz, L. Nilson, H.
Oda, F. Pichaud, J.P. Vincent, and the Bloomington/Kyoto Stock
Centers for flies and other reagents used in this study. We thank the
M. J. Murdoch Charitable Trust for the spinning disk microscope used
for imaging.
This work was supported by the National Institutes of Health [grant
number GM092731 to S.M.P.]. Deposited in PMC for release after
12 months.
Supplementary material available online at
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Drosophila epithelial wound repair 5997
    • Several experimental techniques have been used to analyze collective cell behavior in response to injury. Of these, laser micro-irradiation is readily implemented and allows changes in cell morphology and movement to be monitored with high spatiotemporal resolution (Abreu-Blanco et al., 2012; Antunes et al., 2013). Using this methodology we first characterized quantitatively the dynamic changes of epithelial cell shape and collective responses, which will permit direct comparison with results from numerical simulations (see below).
    [Show abstract] [Hide abstract] ABSTRACT: Epithelial tissues form physically integrated barriers against the external environment protecting organs from infection and invasion. Within each tissue, epithelial cells respond to different challenges that can potentially compromise tissue integrity. In particular, cells collectively respond by reorganizing their cell-cell junctions and migrating directionally towards the sites of injury. Notwithstanding, the mechanisms that define the spatiotemporal scales and driving forces of these collective responses remain poorly understood. To address this we first analyzed the collective response of epithelial monolayers to injury and compare the results with different computational models of epithelial cells. We found that a model that integrates the mechanics of cells at the cell-cell and cell-substrate interface as well as contact inhibition of locomotion predicts two key properties of epithelial response to injury as: 1) local relaxation of the tissue and 2) collective responses involving the elongation of cells (basal and apical regions) and extension of cryptic lamellipodia that extend up to < 3 cell diameters from the site of injury. Our results therefore highlight the integration between junctional biomechanics, cell substrate adhesion and contact inhibition of locomotion to guide the rapid collective rearrangements that are required to preserve the epithelial barrier in response to injury.
    Article · Feb 2017 · Molecular Biology of the Cell
    • The sdt K85 allele [37] was used for evaluation of the function of (mutant) Sdt proteins in vivo by producing germ line clones with the female sterile OvoD technique [38] using FRT19A-OvoD1, hs::Flp (BL#23880). Females of sdt K85 , FRT19A- OvoD1, FRT19A; Ubi::Sdt-GFP were mated with males carrying an FM7-ChFP-fluorescent balancer [39] and Ubi::Sdt-GFP. Embryos which were homozygous for sdt K85 were identified using staining against sex lethal [40] for immunostainings and by sorting against ChFP for cuticle preparations and lethality tests.
    [Show abstract] [Hide abstract] ABSTRACT: In Drosophila, the adaptor protein Stardust is essential for the stabilization of the polarity determinant Crumbs in various epithelial tissues, including the embryonic epidermis, the follicular epithelium and photoreceptor cells of the compound eye. In turn, Stardust recruits another adaptor protein, PATJ, to the subapical region to support adherens junction formation and morphogenetic events. Moreover, Stardust binds to Lin-7, which is dispensable in epithelial cells but functions in postsynaptic vesicle fusion. Finally, Stardust has been reported to bind directly to PAR-6, thereby linking the Crumbs–Stardust–PATJ complex to the PAR- 6/aPKC complex. PAR-6 and aPKC are also capable of directly binding Bazooka (the Drosophila homologue of PAR-3) to form the PAR/aPKC complex, which is essential for apical–basal polarity and cell–cell contact formation in most epithelia. However, little is known about the physiological relevance of these interactions in the embryonic epidermis of Drosophila in vivo. Thus, we performed a structure–function analysis of the annotated domains with GFP-tagged Stardust and evaluated the localization and function of the mutant proteins in epithelial cells of the embryonic epidermis. The data presented here confirm a crucial role of the PDZ domain in binding Crumbs and recruiting the protein to the subapical region. However, the isolated PDZ domain is not capable of being recruited to the cortex, and the SH3 domain is essential to support the binding to Crumbs. Notably, the conserved N-terminal regions (ECR1 and ECR2) are not crucial for epithelial polarity. Finally, the GUK domain plays an important role for the protein’s function, which is not directly linked to Crumbs stabilization, and the L27N domain is essential for epithelial polarization independently of recruiting PATJ.
    Full-text · Article · Nov 2016
    • The first mechanism involves gap closure by cell migration with lamellipodium protrusions, controlled by Rac1 GTPase [51, 46]. The second mechanism involves purse-string closure by assembly and contraction of a multicellular actomyosin belt lining the gap [52, 53]. This process is controlled by RhoA, ROCK, and myosin light-chain kinase (MLCK).
    [Show abstract] [Hide abstract] ABSTRACT: Endothelial hyperpermeability is involved in several critical illnesses, and its regulatory mechanisms have been intensively investigated. It was recently reported that the activation peptide of coagulation factor IX enhances cell matrix and intercellular adhesion. The aim of this study was to investigate the role of activation peptide of coagulation factor IX in intercellular adhesion of endothelial cells and evaluate its effects on endothelial permeability. In the presence of activation peptide, cells spread with lamellipodium-like broad protrusions multi-directionally, increasing the area of adhesion to matrix by 16% within 30 min. In intercellular adhesion, treatment with activation peptide induced overlapping of adjacent cell edges and remodeling of intercellular adhesion sites, with colocalization of the adherens junction proteins VE-cadherin and β-catenin and a marker protein of the lateral border recycling compartment, PECAM. Activation peptide decreased gaps between cells by 66% in cultured endothelial cells and suppressed increased endothelial cell monolayer permeability induced by interleukin-1β in a dose-dependent manner. Treatment with activation peptide decreased eNOS protein expression and altered its subcellular distribution, decreasing intracellular cGMP. An analogue of cGMP suppressed the effects of activation peptide on cell spreading. Additionally, the effect of activation peptide on hyperpermeability was investigated in mice injected with lipopolysaccharide (LPS). Intravenous injection of LPS increased lung weight by 28%, and treatment with activation peptide significantly suppressed the increase in lung weight to 5%. Our results indicate that activation peptide of factor IX regulates endothelial intercellular adhesion and thus could be used in the treatment of vascular hyperpermeability.
    Full-text · Article · Jun 2016
    • The same forces may also modify the " tensegral " stability of the cell, resulting in mechanotransduction [9,10], which in turn control many biological processes. Damaged cells are able to restore the plasmalemma barrier function through addition of membrane material to the plasmalemma surface, dynamic changes in the shape of the plasmalemma [11] and by restoring normal cytoskeletal structures (e.g., via the formation of an actomyosin purse-string121314151617 ). As such, in addition to restoring the barrier function of the cell membrane, single cell wound healing can also be viewed as an attempt at restoring tensegrity to the wounded cell.
    File · Data · Sep 2015 · Molecular Biology of the Cell
    • Damaged cells are able to restore the plasmalemma barrier function through addition of membrane material to the plasmalemma surface, dynamic changes in the shape of the plasmalemma [11] and by restoring normal cytoskeletal structures (e.g. via the formation of an actomyosin purse-string121314151617). As such, in addition to restoring the barrier function of the cell membrane, single cell wound healing can also be viewed as an attempt at restoring tensegrity to the wounded cell.
    [Show abstract] [Hide abstract] ABSTRACT: Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g. microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and effect single-cell repair.
    Full-text · Article · Jul 2015
    • Appropriately staged embryos were fixed, stained, and mounted onto glass slides according to standard procedures (Abreu-Blanco et al., 2012). The following 1° antibodies were used to stain the embryos: rabbit anti-GFP (1:125; Molecular Probes/Invitrogen) and rat anti-DCad2 (1:100; Developmental Studies Hybridoma Bank).
    [Show abstract] [Hide abstract] ABSTRACT: Drosophila immune cells, the hemocyte, undergo four stereotypical developmental migrations to populate the embryo where they provide immune reconnoitering, as well as a number of non-immune related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott Aldrich Syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibits the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex whose activity is needed for this migration, whereas members of the WASH Regulatory Complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex-independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Mar 2015
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