© 2016. Published by The Company of Biologists Ltd.
CONNEXIN 26 GAP JUNCTION COUPLING SELECTIVELY CONTRIBUTES TO
REDUCED ADHESIVITY AND INCREASED CELL MIGRATION
Srikanth R. Polusani*,‡, Edward A. Kalmykov*,‡,
Zucker§,║ and Bruce J. Nicholson*,║¶
Anjana Chandrasekhar *,†, Shoshanna N.
*
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX
78229, USA
§
Current address: Department of Pharmaceutical, Social and Administrative Sciences, School of
Pharmacy, D'Youville College, Buffalo, NY, 14201
‡
These authors contributed equally to the experiments and data analysis and share primary authorship
║ These authors contributed equally to the direction and ideas, and share senior authorship of the
paper.
¶ Author to whom correspondence should be addressed:
Key Words: Gap junctions, connexins, cell motility, cell adhesion
JCS Advance Online Article. Posted on 24 October 2016
Journal of Cell Science • Advance article
Dr Bruce J. Nicholson
Professor and Chair
Department of Biochemistry, MSC 7760
University of Texas Health Science Center at San Antonio
7703 Floyd Curl Drive
San Antonio, Texas 78229-3900
phone: (210) 567-3772
fax:
(210) 567-6595
email: [email protected]
ABSTRACT
Gap junction proteins (connexins) have critical effects on cell motility in many systems, from
migration of neural crest cells to promotion of metastatic invasiveness, Here we show that
expression of Cx26 in HeLa cells specifically enhances cell motility in scrape wounding and sparse
culture models. This effect is dependent on gap junction channels and is isotype specific (Cx26
enhances motility, while Cx43 does not. Cx32 has an intermediate effect). The increased motility
is associated with reduced cell adhesiveness, caused by loss of N-cadherin protein and RNA at the
wound edge. This in turn causes a redistribution of N-cadherin binding proteins (p120catenin and
-catenin) to the cytosol and nucleus, respectively. The former activates Rac-1, which mediates
cytoskeletal rearrangements needed for filopod extension. The latter is associated with increased
expression of urokinase plasminogen activating receptor (an activator of extracellular proteases)
and secretion of extracellular matrix components like collagen. While these effects were dependent
on Cx26 coupling of the cells, they are not mediated by the same signal (i.e. cAMP) by which
INTRODUCTION
The coordination of cell communities is essential for the normal development and function
of tissues in complex eukaryotes. Complementing the myriad of endocrine signaling mechanisms
that help to achieve this, direct exchange of ions, signals and metabolites between cells through
gap junctions is a central component of the integration of cell behavior. Like extracellular signaling
pathways, there is evidence that exchanges through gap junctions shows surprising selectivity. The
connexin composition of the channels [there are 21 connexin genes in humans – (Willecke et al.,
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Cx26 has been shown to suppress proliferation in the same system.
2002)] imparts a surprising diversity in structure and properties (Sosinsky and Nicholson, 2005),
that include single channel conductance [ranging from 15 to over 300pS (Bukauskas and Verselis,
2004, Ek-Vitorin and Burt, 2013) permeability to larger molecules [e.g. nucleotide variants, IP3,
sugars, glutathione, etc. (Bevans et al., 1998, Sneyd et al., 1998, Goldberg et al., 1999) gating
responses (Harris and Contreras, 2014), regulation by different kinases (Lampe and Lau, 2000,
Mitra et al., 2012, Solan and Lampe, 2014) and association with various cytoplasmic and
membrane proteins (Giepmans, 2004, Laird, 2010). In addition, connexins form hexameric
hemichannels (connexons) that, prior to docking, form intercellular gap junction channels, can
mediate release of larger molecules to the extracellular space (Goodenough and Paul, 2003, Saez
and Leybaert, 2014).
Integration of cell behavior is particularly critical during cell migration, whether it is
associated with normal developmental processes or pathological conditions such as metastasis.
Cx43 expression and coupling of neural crest cells has been shown to facilitate the timing of their
migration that is critical to the normal development of the heart (Huang et al., 1998, Francis et al.,
2011). In this case, the C-terminal domain and its interaction with N-cadherin and p120ctn
signaling was implicated, rather than the intercellular coupling function of Cx43 (Xu et al., 2001).
along radial glia during the development of the cortex, although in this case the adhesive role of
the connexins was implicated (Elias et al., 2007, Elias et al., 2010, Valiente et al., 2011) . Changes
in connexin expression have been reported during wound healing of the skin (Coutinho et al., 2003,
Churko and Laird, 2013), where expression of Cx43 was shown to be downregulated while Cx26
was shown to be upregulated in the epidermal layers during wound healing (Kretz et al., 2003,
Goliger and Paul, 1995).
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Both Cx43 and Cx26 expression have been shown to be important for migration of cortical neurons
Connexin expression has also been associated with increased motility and invasiveness of
tumor cells, reviewed in (Defamie et al., 2014, Kotini and Mayor, 2015) . The invasion of HeLa
cells into heart fragments was enhanced by expression of Cx43, Cx31 or Cx40 (Graeber and
Hulser, 1998). Several in vivo assays also linked Cx expression with increased invasiveness of
malignant glioma cells (Lin et al., 2002) and hepatocellular carcinomas (Ogawa et al., 2012). In
malignant melanoma cells, a change in cadherin expression during tumor progression helped
promote coupling with dermal fibroblasts and increased invasiveness (Hsu et al., 2000).
Expression of Cx43, and its associated coupling, increased the migration rate two-fold in an assay
of breast cancer cell invasion into underlying endothelial cells (Pollmann et al., 2005). Homotypic
Cx43-mediated GJIC in early and advanced metastatic prostate carcinoma cells also enhanced their
migration rate (Miekus et al., 2005). There have been some contrary findings where Cx43 or Cx26
(Defamie et al., 2014) expression has been linked to suppressed motility or metastasis. However,
most studies, including clinical correlations of multiple cancers [skin (Kamibayashi et al., 1995)
, breast (Kanczuga-Koda et al., 2006), prostate (Tate et al., 2006), and colorectal (Ezumi et al.,
2008 )], support a role for increased Cx43 or Cx26 functional expression being linked to enhanced
motility and invasiveness of tumor cells.
can enhance motility in a well-established cancer cell line (HeLa cells) where the connexin
composition can be precisely controlled. This allowed us to assess if the effects on cell motility
are connexin-specific, as well as to rigorously test which specific function of connexins are
important in regulating motility. This strategy has allowed us to identify a signaling cascade that
links enhanced gap junction coupling to facilitation of the motile phenotype that is likely to be
useful in understanding the role of gap junctions in metastasis and other processes in development.
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The current work addresses the issue of the underlying mechanisms by which gap junctions
RESULTS
In order to distinguish the mechanisms by which connexins regulate proliferation and
motility, we utilized the same HeLa cell lines described in (Chandrasekhar et al., 2013). Nonclonal, and in some cases clonal, populations of HeLa cells were prepared expressing Cx26,
Cx43 and Cx32, as well as variants of Cx26 that form normal gap junction structures, but fail to
form functional channels [Cx26T135A – (Beahm et al., 2006)] or only form functional
hemichannels [Cx26R75Y – (Deng et al., 2006)]. These cell lines are extensively characterized
in (Chandrasekhar et al., 2013) for comparable levels of protein expression, appropriate cellular
localization, as well as both gap junction and hemichannel function. The former was assessed
using calcein dye transfer in a parachuting assay and the latter assessed by uptake of Lucifer
Yellow (Table IS). Note that dye uptake was relatively low, as experiments were done in normal
extracellular Ca++ (a known blocker of hemichannels) to emulate the conditions of the
subsequent motility assays.
intercellular coupling.
The effect of Cx expression on cell motility was assessed using a scrape wounding assay
of the cell monolayer. Figure 1A shows the time course of “wound healing” in HeLaWT, and
representative clones of each connexin transfectant. While the cell densities of the monolayers
and width of the initial streaks were comparable in all cultures, rates of wound healing differed
dramatically. After 48 hours, the HeLaWT and HeLa43 cells had only minimally filled the wound,
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HeLa 26 expression selectively enhances wound healing in a manner that is dependent on
while HeLa26 cells had almost completely filled in the wounded area. HeLa32 cells had an
intermediate phenotype. Point mutants of Cx26 that have lost gap junction (Cx26R75Y) or both
gap junction and hemichannels activity (Cx26T135A) failed to show any enhancement of
migration over the WT HeLa cells (Fig. 2), demonstrating the requirement for intercellular
coupling.
Metamorph software allows tracking of individual cell movements over time (Figure 1B),
which confirmed the dramatic differences in migration rates of Cx26, 32 and 43 expressing cells.
Cx26 expressing cells moved 2 times faster into the wound (defined as direction “X”) than HeLa
, or Cx43 and non-functional Cx26 expressing cells, and 1.5 times faster than Cx32 expressing
cells (Figure 1C). The directionality of movement was enhanced to an even greater degree, with
a three-fold increase in the ratio of movement into the wound (X direction) compared to parallel
to the wound edge (Y direction) in HeLa26 compared to all other transfectants other than HeLa32
(where the increase was only two fold). Real time video imaging of the wound healing (Video 1 see Methods for access address) reveals that HeLaPar and HeLa43 cells migrate as sheets, whereas
HeLa26 cells tend to separate out and migrate as single cells, or clusters of cells, at the wound
edge, and show much greater filopod extending activity. Cells expressing the channel impaired
Using several individual HeLa26 transfected clones, we further compared the rates of
wound healing with the levels of Cx26 expression and coupling. Three clones (C, M, and 11)
showed similar coupling levels and wound healing rates, measured as days to heal the wound
(Figure 2A: filled and open bars, respectively). However, in two clones (12 and 16) with notably
lower coupling levels, the days taken to heal the wound increased as coupling decreased. This was
also true for the two Cx26 mutants that fail to form gap junctions (Cx26R75Y and T135A). While
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Cx26 mutants migrate as sheets, like HeLaWT cells.
there was a correlation between level of Cx26 coupling and rate of wound healing, there was no
correlation with levels of protein expression.
Increased wound healing in HeLa26 is not a product of enhanced mitosis.
Increased rates of wound closure could also be influenced by rates of cell division, although
this could not explain the tracking data we obtained. Nonetheless, this was directly tested by
measuring levels of bromodeoxyuridine (BrdU) incorporation at both the leading edge of the
wound as well as in the confluent monolayer distal to the wound in each HeLa culture (Figure 1D).
The percent of mitotic cells (BrdU positive cells) distal to the wound site was similar for all cells
studied, consistent with the similar growth of the different Cx transfectants in 10% serum [Cx26
only affected growth in 1% serum (Chandrasekhar et al., 2013)]. By contrast, at the leading edge,
HeLa26 cells showed a decrease in mitotic cells compared to the distal region, whereas both
HeLa43 and HeLaWT showed an increase in BrdU labeling. The mitotic index of HeLa 26 at the
leading edge was approximately 2/3 of that measured for HeLa43 and WT cells, indicating motility
Co cultures of connexin-expressing cells show the influence of heterotypic coupling.
The differential effects of Cx26, 32 and 43 on the migration of HeLa cells indicates that
the intercellular channels composed of the different connexin isoforms may preferentially pass
distinct signals, with Cx26 most effective, and Cx43 least effective in enhancing motility. This
leads to a testable prediction that, should heterotypic Cx26/32 channels be intermediate in
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and mitotic activity at the wound edge were inversely correlated.
permeability between Cx26 and Cx32 homotypic channels, then in mixed cultures, HeLa26 cells
may be able to influence the motility of co-cultured HeLa32. Also, since Cx26 and Cx43 cannot
form heterotypic gap junctions, one would predict that these cells would have no influence on one
another’s motility.
To test this, we performed migration assays on co-cultures of HeLa26/HeLa43 and
HeLa26/HeLa32 plated at 1:1 ratios, where each cell type was differentially labeled with a
lipophilic green or red fluorphor. As predicted above, when HeLa26 and 43 cells were mixed, and
subjected to wounding, the HeLa26 cells filled the wound first, leaving the HeLa43 cells behind
(Figure 3A, top). In contrast, when monolayers of mixed HeLa26 and HeLa32 cells were wounded,
HeLa32 cells migrated along with the HeLa26 cells, filling the wound equally (Figure 3A, bottom).
This is most evident in the time-lapse videos at (Video 2 and 3 – see Methods for access address)
By tracking the motility of individual cells as shown above, we can quantitatively compare
the rates of movement of each cell type into the wound (Figure 3B). This clearly demonstrates that
the rate of movement of HeLa26 cells remained constant under homotypic or either heterotypic
scenario (green bars). Similarly, HeLa43 cell motility was much lower, and was not influenced by
heterotypic culture with HeLa26 cells (red bars-right row), consistent with the inability to form
heterotypic cultures with HeLa26 compared to their motility in homotypic culture, showing
comparable motility to HeLa26 (red bars-middle row). Thus, heterotypic Cx32/26 channels appear
to be as effective in propagating motility enhancing signals as Cx26 channels alone. This also
demonstrates that the motility behavior is not dictated by the connexin expressed by a specific cell,
but by what information is exchanged with its neighbors. The failure of HeLa26 to influence
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Cx43/26 heterotypic channels. In contrast, the motility of HeLa32 cells almost doubled in
HeLa43 motility, also demonstrates that the signals are not transmitted extracellularly through the
media.
Cx26 induced increased migration is not just a response to wounding, but a general
enhancement of motility.
While the wound healing assay is a useful method to quantitate motility, it can include
specific responses to signals associated with the wounding process itself. To determine if Cx26
effects on motility are specific to this condition, we also compared motility of HeLaPar, HeLa26
and HeLa26R75Y in sparse cultures by tracking individual cell movements. While under these
conditions, there was no directionality to cell movement, the HeLa26 cells clearly showed much
more motility and extension of filopodia (Figure 4A; also see Video 4 – see Methods for access
address). When the tracks of multiple cells were followed, HeLa26 cells traveled approximately
7-fold further in a given timeframe than HeLa Par or HeLaR75Y cells (Figure 4B). This dramatic
increase in the movement in sparse cultures was surprising given our demonstration above that the
enhanced motility in the wound healing assay was dependent on GJIC. It has generally been
assumed that intercellular coupling is minimal in sparse cultures, conditions in which connexins
through hemichannels, as the Cx26R75Y mutant, which forms hemichannels as, or more
efficiently than WT, had no effect on motility.
Thus, we directly assessed coupling in these sparse cultures by labeling a subpopulation of
HeLaWT or HeLa26 cells with DiI and also pre-loading them with a calcein-ester, before mixing
them with an excess of unlabeled cells and plating under sparse conditions. After allowing time
for the cells to adhere (5-6 hours), cells were sorted and the % of cells now showing Calcein
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predominately form hemichannels. However, in this case the effects on motility were not mediated
labeling alone, with no DiI label (i.e. they received calcein through gap junctions from the original
double labeled cells) was determined (Figure 4C – lower right quadrant). Surprisingly, the data
revealed that the sparse cultures show a high degree of cell communication. While HeLaWT cells
showed 2.5% and 1.2% of DiI negative cells picking up calcein in confluent and sparse cultures,
respectively, In contrast, HeLa26 cells showed 15.6% and 8.8% of DiI negative/Calcein positive
cells under the same confluent and sparse conditions, respectively. This result demonstrates that
the transient cell contacts that are seen between these mobile cells in the time lapse movies (Video
4) allow cell communication to occur 50% as effectively as happens in confluent monolayers. This
also demonstrates that increased migration of HeLa26 is an inherent property of the cells and not
a product of wounding.
N-cadherin is necessary and sufficient to modify migration of HeLa cells,
In searching for clues as to what the downstream targets of these intercellular signals might
be, a comparison of cell behaviors during both wound healing and sparse culture assays (see
Videos 1 and 4) revealed much reduced cell-cell adhesiveness among HeLa26 compared to
parental and other transfectants. Not only do HeLa26 cells separate out from the advancing wound
after cell division was very short lived. Hence, we examined expression of N-cadherin, the primary
adhesion molecule expressed in HeLa cells. Immunofluorescence showed a loss of N-Cadherin
and relocation to the cytosol at the wound edges of HeLa26 compared to HeLaWT or all other
HeLa transfectants (HeLa43 shown - Figure 5A). This phenotype was evident in all individual
HeLa26 and 43 cells (Fig. 1S). The reduction of N-Cadherin in HeLa26 was also seen in Western
blots (Figure5B), and at the RNA level by quantitative RT-PCR (Figure 5C) from cultures that
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edge much more frequently, in sparse cultures HeLa26 cells do not form colonies, and contact
were “stamp wounded” to create multiple wounding interfaces (see Methods). Comparisons of Ncadherin levels in unwounded and wounded cultures demonstrated that Cx26 decreased Ncadherin expression compared to that in HeLa43 and WT cells by 3-4 fold in confluent cultures,
and to a much greater extent in wounded cultures. HeLa32 cells also showed a similar pattern of
decreases, but to a lesser extent than in HeLa26 (Fig 2S).
The functional importance of N-Cadherin in regards to motility was demonstrated by
siRNA knockdown in HeLa43 (Figure 5D) and HeLaWT cell lines (Fig 2bS) to levels similar to
those seen in HeLa26. This resulted in a significant increase in the migration of these cells to rates
close to that of HeLa26 (Figure 5E). Scrambled siRNAs had no effect on motility. Conversely,
exogenous expression of N-cadherin in HeLa26 to levels comparable to that seen in HeLaWT,
(Figure 5F) significantly decreased the rate of migration of these cells compared to cells transfected
with empty-vector alone, although not to the level of HeLaWT cells (Figure 5G). These studies
demonstrate that N-cadherin is both necessary and sufficient to modify migration of HeLa cells,
although it appears that it cannot fully account for the Cx26 motility phenotype.
migration.
The cytosolic domains of Cadherins have been shown to associate with the armadillo
family proteins, including β-, γ-, and p120 catenins (p120ctn) (Reynolds et al., 1994). Loss of Ncadherin would result in the release of these components into the cytoplasm, where they have been
shown to play other roles. Specifically, p120ctn-RhoGTPase has been implicated in the
modulation of the actin cytoskeleton, leading to branching that increases filopodial/lamellepodial
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Redistribution of N-Cadherin binding proteins in HeLa26 is also linked to increased
activity and migration (Anastasiadis and Reynolds, 2000) (Noren et al., 2000) , (Reynolds et al.,
1996);
(Grosheva et al., 2001). By contrast, β-catenin has been extensively studied as a
transcription factor that typically stimulates oncogenic gene expression (Behrens, 2000).
Although N-cadherin levels are much reduced in HeLa26, total p120 catenin levels, which
show multiple bands on gels due to posttranslational modification, were similar in all the clones
(Fig 6A). However, with the loss of the N-cadherin tethering it to the membrane in HeLa26 cells,
the levels of cytosolic p120ctn were significantly elevated (Figure 6A). This cytosolic
displacement of p120ctn caused activation of Rac to its GTP-bound form in HeLa26 cells, as
shown by immunoblot (Figure 6B). The functional importance of Rac activation in migration was
tested by siRNA knock down of Rac in HeLa26 (Figure 6C), which resulted in a significant
decrease in migration rates to levels indistinguishable from HeLa43 (Figure 6D).
We also looked at the localization of β-Catenin, another major N-cadherin binding protein
at the regions distal to, and at, the edge of the wound. HeLa and HeLa43 cells showed β-Catenin
colocalization with N-Cadherin at the cell membrane, both distal and proximal to the wound (Fig.
5A). In contrast, HeLa26 cells showed this same co-localization in the monolayer distal to the
1S). Since β-catenin is a major transcriptional co-activator, we tested several known transcriptional
targets of β-catenin. Two such targets associated with the motile phenotype, the urokinase
plasminogen activator receptor (uPAR) (Figure 6F) and Collagen IV (Fig 6G), showed selective
increases in both RNA and protein levels in HeLa 26 wounded cultures. Another extracellular
matrix protein (ECM), fibronectin, also showed an increase in HeLa26, but in this case HeLa43
also showed an increase.
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wounds, but a redistribution of β-Catenin to the nucleus at the wound edges (Figure 5A, 6E and
uPAR plays a central role in tumor development through control of cytoskeletal dynamics,
cell adhesion and extracellular matrix (ECM) integrity (Blasi and Carmeliet, 2002), while collagen
IV and fibronectin are important components of the ECM needed to support cell motility in culture.
Consistent with the latter, the migration rate of HeLaWT and HeLa32 cells in the wound healing
assay increased on plates pre-coated with collagen, while this had little effect on the HeLa26 cells
(Fig 4S), presumably because they make their own collagen.
DISCUSSION
There have been several reports linking connexin expression to modulation of cell motility
(Huang et al., 1998, Xu et al., 2001, Francis et al., 2011, Churko and Laird, 2013, Elias et al., 2007,
Elias et al., 2010, Valiente et al., 2011). In cancer, enhanced connexin expression, or specifically
surface localization, have been correlated with increased metastasis (Defamie et al., 2014, Kotini
and Mayor, 2015), which typically also involves an enhanced migratory phenotype. Using a
enhanced when the cells are coupled by Cx26. This effect requires formation of intercellular
channels, unlike some neuronal systems reported previously where the mechanisms were linked
to the structural characteristics of gap junctions, or their role in adhesion (Xu et al., 2001, Elias et
al., 2007). It was further shown to be dependent on the connexin isoform, as Cx43 coupling did
not change motility and Cx32 had an intermediate effect.
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controlled exogenous expression system, we now show that the motility of HeLa cancer cells is
A second major finding is that Cx26 intercellular coupling mediates enhanced motility
through a significant loss of cell adhesion. Several studies in the past have reported that cell
adhesion enhances, or is required for, intercellular coupling (Keane et al., 1988, Jongen et al.,
1991, Musil et al., 1990, Zhu et al., 2010) . However, this is the first study to show that gap
junctional coupling can have a reciprocal effect on adhesion. It is particularly intriguing that this
is not due to the close physical associations between the components, but is caused by exchanges
of intercellular signals specific to Cx26 that cause a loss of N-cadherin transcription (or a
destabilization of its mRNA).
The HeLa system recapitulates many of the early steps of metastasis, with an up-regulation
of coupling (induced exogenously in this case), a drop in cellular adhesiveness and enhanced
motility. As HeLa cells have lost the E-cadherin gene, adhesion is mediated by N-cadherin. Figure
7 summarizes the mechanism whereby the down-regulation of N-cadherin in HeLa26 cells leads
to decreased adhesivity, causing release of N-cadherin binding proteins p120ctn and β-catenin to
the cytosol and nucleus, respectively where they activate targets to promote enhanced migration.
In terms of these downstream effects, p120ctn overexpression in fibroblasts has previously
adhesion formation, and augmented migratory ability (Grosheva et al., 2001). This has been
attributed to potent inactivation of RhoA (Anastasiadis and Reynolds, 2000, Noren et al., 2000)
and/or activation of Rac1, which we directly demonstrate in this study, and Cdc42 (Noren et al.,
2000, Grosheva et al., 2001) . p120ctn has also been shown to stabilize microtubules important for
directional cell migration in a cadherin independent manner (Ichii and Takeichi, 2007).
One interesting consequence of the loss of N-cadherin induced by Cx26 coupling is that,
as Cx26 transfectants migrate, the cells at the leading edge are sparsely populated and are often
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been shown to induce a dramatic actin branching phenotype, with increased contractility and focal
not in direct contact with adjacent cells (Video 1.). This can lead to loss of coupling at the leading
edge of cell migration, accompanied by a loss of connexin plaques. It seems paradoxical that GJIC
is necessary for cell migration, yet it may be disrupted among the most motile cells. Two
possibilities exist to explain this observation. First, Cx26 GJIC may serve to “prime” the actions
of the cells at the wound edge by inducing the initial loss of N-cadherin. This triggers the cascade
of events that promote motility, after which continued coupling may not be needed. Second,
despite the general lack of contact, some coupling may still be retained. This is consistent with our
observations in sparse cultures, where we demonstrated that even in highly motile cells that show
minimal adhesion, and only transient contacts, coupling can still be quite effective (Figure 4C).
This observation is highly significant, as it suggests that during cell migration in development and
tumorigenesis, transient contacts with the surrounding tissue may be critical in guiding motility,
or in the extravasation of tumor cells that make transient contacts with endothelial cells of blood
vessels or lymphatics.
As yet, we have not identified the initial intercellular signal that mediates the loss of Ncadherin, and ultimately enhanced modulation, induced by Cx26 coupling. The challenge, like
all gap junction regulated processes, is the diversity of possible candidates, notably all signaling
permeant (Sneyd et al., 1998), which affects migration by activating the Rac-cdc42 pathway (Coon
and Herrera, 2002); Calcium, another junctional permeant that has been shown to regulate motility
through protein kinase C (Etienne-Manneville and Hall, 2001). Even larger molecules, like
microRNAs, have also shown to pass through gap junctions (Zong et al., 2015), and can regulate
both motility and adhesion [e.g. miR-124 (Zhang et al., 2015)]. Of particular interest is cAMP, the
intercellular signal that mediates growth suppression in HeLa cells, which is also shown to be
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molecules under 1000 in MW. Some notable candidates could be: IP3, a known junctional
specific to Cx26 (Chandrasekhar et al., 2013). cAMP is also known as a regulator of cadherin
expression through cAMP response elements (Pon et al., 2005).
To test this, we monitored the spatial distribution of cAMP by antibodies directed to the
activated form of PKA, which we showed to reflect the actual concentration of cAMP in the cells.
This revealed a marked upregulation of PKA activity at the wound edge in HeLa cells (Fig. 8A)
that was eliminated in HeLa26 cultures (Fig 8B), consistent with a redistribution of cAMP. The
distribution of PKA activity is broadened in HeLa26 cells at the wound edge, presumably because
these motile cells are minimally in contact (Fig. 8B – yellow bars). However, the difference in
PKA activities at the wound edge and the monolayer distal to the wound is also eliminated in
HeLa43 cultures (Fig 8C), although in this case the distributions in each population remain tight
as all the cells retain coupling. However, since migration and N-cadherin levels are not affected in
HeLa43 cells, the redistribution of cAMP cannot be the determining signal for motility. Hence,
the suppression of cell growth and promotion of motility, both induced selectively by Cx26, are
mediated by different signals. This opens possibilities for targeting connexins therapeutically in
cancer, and other areas, by differentially affecting growth and motility effects.
Videos available in File Share:
Video 1: https://figshare.com/s/bbd9ddb582b1a0f0637c (download to see all frames)
Video 2: https://figshare.com/s/c05749d9046bdffdc077
Video 3: https://figshare.com/s/0aaa849fd009bec0209f
Video 4: https://figshare.com/s/addf823e0d98c4759a1f (download to see all frames)
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MATERIALS AND METHODS
Cells and transfections
Wild type HeLa cells were a gift of Dr. Klaus Willecke (University of Bonn, Germany). Each WT
connexin was inserted into a pIRES hygro vector (Clontech, Palo Alto, CA), and following
lipofection, (Lipofectamine plus, Invitrogen, Carlsbad, CA) clones were selected using 400 µg/ml
hygromycin (Sigma, St. Louis, MO). Cx26T135A or Cx26R75Y DNA was inserted into a
pIRESpuro3 vector, and pools of transfectants were selected with 1 µg/ml puromycin (sigma, St.
Louis, MO) and maintained in 0.5 µg/ml puromycin. Cells were tested for protein expression
(Western blot and immunofluorescence) and functional coupling (dye preloading assays). Cx26
clones C, M and 11 were pooled in equal ratios for functional and migration analyses. siRNA for
N-Cadherin (Dharmacon, Lafeyette, CO), or Rac (Pierce, Rockford, IL) were used to knock down
their respective RNAs by transfection using 100-200 nM siRNA with Dharmafect reagent and
Lipofectamine respectively. N-Cadherin was overexpressed in HeLa26 cells by transfection of NCadherin in a pCMV myc vector (Origene, Rockville,MD) using lipofectamine (Invitrogen,
Carlsbad, CA).
Cells were plated onto 12 mm round coverslips and streaked with a pipette tip after they reached
confluence.
After 24 hours, the cells were fixed (2% paraformaldehyde for 20 minutes),
permeabilized (1% Triton X-100 in PBS for 20 minutes), and blocked (0.1M Glycine/0.5% BSA
in PBS for 1 hour at 370C) prior to labeling with the relevant primary (Zymed, Invitrogen,
Carlsbad, US, 1:100), and secondary antbodies (goat anti-rabbit conjugated to Alexa fluor 488,
Molecular Probes, Eugene, OR). Slides were viewed at 60X and Zoomed 3X, with an Olympus
FV-500 Confocal Laser Scanning Microscope in the Core Optical Imaging Facility at UTHSCSA.
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Confocal imaging for gap junctions
Confocal images were collected using an Olympus Fluoview 500 laser scanning system mounted
on an Olympus IX-81 microscope using a 60X Uplan Apo, NA 1.4. Alexa-488 was excited by the
488 nm line of an Argon laser and imaged through a 505-525 nm bandpass filter. Images shown
were assembled from a Z stack taken at 0.5 mm intervals.
Dye transfer and uptake assays
Coupling was assayed using the pre-loading method of fluorescent dye transfer (McNeil et al.,
1984). The donor cells were incubated with two fluorescent dyes: calcein AM (5mM) and DiI
(10mM) (Molecular Probes, Eugene, OR), for 20 min in isotonic glucose. These pre-loaded donor
cells were then plated with unlabeled cells at a ratio of 1: 72 and allowed to settle for 5-6 hours.
The percent coupling of each clone was determined as the percentage of preloaded cells passing
Calcein to at least one receiver cell (donors identified by DiI). Data was obtained from at least
two independent experiments, with at least 100 donors. For assay of hemichannel function, cells
were grown in 24 well plates, washed with PBS and mechanically stimulated by dropping 0.5 ml
of PBS in all four quadrants of the well. Immediately, 2% Lucifer Yellow and 2% Rhodamine
washed 5-7 times and dye uptake was observed. Images were photographed with a Zeiss Axiovert
35 photomicroscope, equipped with UV epifluorescence to measure green (Lucifer yellow –
connexin channel uptake) and red (DiI – uptake by damaged cells) fluorescence. Data was
recorded on an AxioCamMR CCD camera and stored digitally using Axiovision software.
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dextran (Molecular Probes) was added to the media and incubated for 10 min. The cells were
Cell migration assays
HeLaWT and transfectants were plated in DMEM + 10% FBS at 4 x 105 per 35 mm tissue culture
dish (Becton Dickinson, Franklin Lakes, NJ) on duplicate plates. 48 hours after plating, a time in
which all cell lines achieved confluence, the plates were streaked with a micropipette tip. After
rinsing off released cells, Photos were taken in 3 regions along the streak at time 0, and every 24
hours for 3 days. For each clone, the time to fill the wound was determined as the point at which
the wound was completely filled with cells. The percent of the wound filled was determined by
measuring the area of the streaked wound at day 0, and counting the number of cells that filled that
area before, and at various times after, wounding. The percent wound filled was the ration of these
numbers. All experiments were done in triplicate These studies were supplemented with real time
in vivo migration videos over the course of 36-48 hr. time period using a Nikon TE microscope
and Metamorph imaging analysis.
Video microscopy and cell tracking
A confluent monolayer of cells in a 60mm plate were wounded with a pipette tip and placed in a
CO2, humidity and temperature controlled chamber attached to a Nikon TE 2000 microscope.
time-lapse stack was created. The cell tracking app module from Metamorph software was used to
calculate the velocity of migration, distance travelled in a defined direction (X – perpendicular to
the wound, Y – Parallel to the wound), and directionality (X/Y) of each cell type. Similarly, the
particle track feature of the software was used to track individual cells using the stack of the images
generated to calculate the velocity and directionality of movement in sparse cultures. In both
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Images of migrating cells were taken over time (1 image/6 minutes) for 48 hours and a user defined
wounded and sparse cultures, at least 10 cells were tracked in each field, and all experiment were
done in triplicate.
Bromodeoxyuridine labeling
The same wounding experiment described above was performed on cells grown on 22 mm square
coverslip (Corning, Acton, MA) At time 0, 1 day or 2 days after wounding the coverslips were
incubated in a freshly prepared 20 µM BrdU (Sigma, St. Louis, MO) in PBS for 30 minutes at
370C. After rinsing, the cells were fixed in 2% paraformaldehyde (Sigma, St. Louis, MO) for 20
minutes, permeabilized with 0.25% triton (Sigma, St. Louis, MO), denatured in 4 M HCl (Fisher
Scientific, Springfield, NJ) for 30 minutes, and after a 5 minute block with 2% BSA (Sigma, St.
Louis, MO) in PBS, exposed to BrdU primary monoclonal antibody (Thermo-Fisher, Waltham ,
MA) at a 1/50 dilution for 2 hours in blocking buffer, followed by anti-mouse Alexa 488
(Molecular Probes, Eugene, OR) for 30 minutes at a dilution of 1/200 in blocking buffer.
Coverslips were viewed at 40X on an Olympus BX51 microscope equipped with a Sensicam CCD
camera. Five fields were photographed for each coverslip including the leading edge (up to 20
cells in from the edge) and a region distal to the wound (approximately 300 cells from the edge).
to the total number of cells per field as determined from phase contrast micrographs.
Co-culture migration cell labeling
HeLa26, HeLa32 and HeLa43 cells were suspended at a density of 1 x 106 /ml in serum free culture
medium. 5 μL of the cell labeling solution (Vybrant Cell-labeling solutions, Molecular Probes,
Eugene, OR), DiO for HeLa26, and DiI for HeLa32 and HeLa43, was added per ml of cell
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The % of BrdU labeled cells was obtained from ratios of the number of fluorescent cells per field
suspension. The treated cells were incubated for 10 mins at 37oC. The labeled cells were
centrifuged at 1500 rpm for 5 mins at 37oC. The cells were resuspended and plated at a density of
1:1, before growing to confluence and performing the wounding assay described above.
Dye transfer in sparse cultures
The donor cells were incubated with two fluorescent dyes: calcein AM (5mM) and DiI (10mM)
(Molecular Probes, Eugene, OR), for 20 min in isotonic glucose. These pre-loaded donor cells
were then plated with unlabeled cells at a ratio of 1: 6 and allowed to settle for 5-6 hours. The cells
were then trypsinized and a FACS sort was performed to separate donor cells (having DiI and
Calcein) from acceptor cells having only calcein on a BD FACS Calibur instrument, with analysis
by the Cell Quest software.
Stamp Wounding system
we used a stamp-wounding system that generated plates of cells where the majority of the cells are
involved in wound healing. The stamp-wounding device is a circular contoured “stamp, the same
diameter as a culture plate, that denudes cultured cells in circular patterns, with intervening strips
of intact cells. These remaining cells migrate and proliferate in near synchrony to fill the wounds
(Lan et al., 2010).
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In order to be able to biochemically analyze (by Western or PCR) cells involved in wound healing,
Western Blots
Total cell lysates were prepared in RIPA buffer (PBS, 1%NP40, 0.5% sodium deoxycholate, 0.1%
SDS, 10 mg/ml PMSF, 10 µg/ml Na3VO4, aprotenin, leupeptin, pepstatin; 1ml/100 mm dish).
Lysates were passed through a needle 18 times to shear the DNA and spun at 15000 rpm for 20
minutes. Protein estimations were performed on the supernatant (MicroBCA protein assay kit,
Pierce, Rockford,IL) with BSA as a standard. 20 µg of protein per sample was separated on a 12%
SDS polyacrylamide gel and electro-blotted onto Imobilon-P membranes. The blots were probed
with monoclonal or polyclonal anti-Cx26, 32 and 43 antibodies (Cell Signaling Technology,
Danvers, MA), followed by a secondary antibody conjugated to HRP (cell signaling) and then
ECL plus reagent (Amersham Biosciences) prior to exposure to X-Ray film (Kodak). The blots
were stripped (100mM 2-mercaptoethanol, 2% w/v SDS, 62.4 mM Tris-HCl, pH 6.7) for 30
minutes at 500C with mild agitation and re-probed with actin antibody (Sigma, St. Louis, MO).
Cell lysates from migrating cells were prepared 24 or 48 hours after stamp wounding confluent
monolayers. Antibodies for N-cadherin (Zymed, San Francisco, CA), p120 Catenin (Santa Cruz
Biotechnology, Santa Cruz, CA) and Rac1 (Pierce, Rockland, IL) were used according to
RTq-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,CA) according to the
manufacturer’s instructions. 1 microgram of total RNA was reverse transcribed using the Taqman
reverse transcription reagents from Applied Biosystems (Foster City, CA). 1 microliter of the
reverse transcription reaction mixture was analyzed by real time quantitative PCR (RTqPCR) with
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manufacturer’s instructions.
SYBR green mix using an ABI Prism 7700 sequence detection system (Applied Bio Systems).
The mRNAs measured were normalized with respect to 18s rRNA. Primers were designed using
primerquest software from Integrated DNA technologies. uPAR forward primer, 5CATGTCTGATGAGCCACAGG-3’,
CTGGAGCTGGTGGAGAAAAG-3’,
and
uPAR
N-Cadherin
reverse
forward
primer,
5’-
primer
5’-
CAGGGAATCCCCCCAAGT-3’ and reverse primer 5’-CATTGGAGTCACATTGGCAAA-3’,
were used.
Rac-GTP pull-down assay
Rac GTP was detected using the Active Rac1 Pull-Down and Detection kit (Pierce Biotechnology,
Rockland, IL). Briefly, cells were collected 1 day after stamp wounding using the
Lysis/Binding/Wash buffer and centrifuged at 16000xg at 4 0 C. The supernatant is then placed on
immobilized Glutathione swellGel discs containing the GST-human PAK1-PBD in a spin cup.
After incubation at 40 C for 1 hr., the spin cup with collection tube is centrifuged at 7200 x g for
10-20 seconds, removed and washed before eluting the protein with sample buffer containing β-
Anti-Rac 1 antibody (Pierce Biotechnology, Rockland, IL).
Statistics
Pairwise comparisons to assess significance of differences shown throughout were performed
using unpaired, two-tailed Student T-tests through Prizm software. Numbers of measurements and
experimental repeats are described in each figure, or the Methods.
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mercaptoethanol and SDS. The sample is then electrophoresed and gel probed for Rac-GTP using
Table 1: Gap Junction and Hemichannel Function in HeLa cell transfectants
Cell Type
% coupled
HeLaWT
HeLa43
HeLa32
HeLa26
HeLa26 T135A
HeLa26 R75Y
0
95 ± 1.3
96 ± 2.0
93 ± 0.7
0
0
% First order
coupling
0
73 ± 10
80 ± 9
83 ± 8
0
0
% Dye uptake
0
ND
ND
3 ± 1.7 *
0
6 ± 1.2
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ND = not determined
*primary uptake only measured (not spread through gap junctions)
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FIGURES
Figure 1: Cx26 expression increases HeLa cell migration in wound healing assay.
Migration rates of HeLa parental cells (par) and HeLa cells transfected with different connexins
were measured in a scrape wounding assay over 2 days
(A) Cx26 expression induced the most rapid filling of the wound, Cx32 showed an
intermediate rate, while Cx43 and a Cx26 mutant (R75Y) that forms hemichannels but no
gap junctions had no effect on rate of wound healing (also see Video 1).
(B) Individual cell movements can be tracked by the Metamorph software using fluorescently
tagged cells (white traces).
(C) This provided, direct measures or migration rates into the wound (X direction in pixels
traversed in 24 hours at 100X – filled bars) as well as directionality (X/Y or the ratio of
movement perpendicular to and parallel to the wound – open bars). HeLa26 cells show
significant increases in both measures compared to HeLaWT. (* = p<0.001; n = 10 cells
each in 3 experiments).
(D) BrdU incorporation as a measure of cell proliferation, showed no differences between
revealed an increase in HeLa parental and HeLa43, compared to a decrease in HeLa26
(filled bars). (* = p<0.05; n = 100 cells counted in 5 fields for each condition).
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HeLaWT and Cx transfectants in the monolayer distal to the wound (clear bars), but
Figure 2: Coupling increases HeLa26 cell migration.
The coupling levels (% of first order neighbors coupled) of various HeLa clones (filled bars) were
compared to the rate of wound healing (days to heal wound). On the left, a comparison of different
connexins showed that all induce similar levels of coupling, but Cx43 did not influence the time
the time to close the wound to 2 days. A comparison of several HeLa26 clones that show variable
levels of coupling (C, M, 11, 12, 16), as well as two Cx26 mutants (T135A and R75Y) which fail
to form channels, showed that reduced coupling levels correlate with increased time to heal the
wound.
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to close the wound (5 days), Cx32 caused some decrease (to 3 days) and Cx26 maximally reduced
Figure 3: Selective heterotypic junctional coupling can also enhance motility
(A) Scrape wounding of 1:1 co-cultures of HeLa26 cells (labeled green), with HeLa43 or
HeLa32 cells, (labeled red) were imaged at 0 (left) and 36 hours after wounding (right).
In the case of the HeLa26/43 co-cultures (upper right), where Cx26 and 43 do not form
heterotypic gap junctions, the HeLa26 cells (green) clearly separate and migrate more
rapidly into the wound than the HeLa43 cells (red) [also see Video2] By contrast,
red cells fill the wound equally, presumably due to Cx32/26 heterotypic coupling (Video
3).
(B) Tracking the motility rates of individual cells into the wound (distance moved into the
wound (X axis) in pixels) in homologous (front row) and heterologous cultures confirms
that HeLa26 cells migrate at similar rates in homotypic and both heterotypic cultures
(green bars). HeLa43 cells migrate at the same rate when alone as when mixed with
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HeLa32 cells (lower right) migrate at similar rates to HeLa26, such that both green and
HeLa26 (red bars in right row), but. HeLa32 cells show enhanced migration compared to
homotypic cultures when in co-culture with HeLa26 (* p < 0.001; n = 3), moving at rates
similar to the HeLa26 cells (red bars – middle row). 10 cells were tracked in 3
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independent experiments. Small boxes on top of each bar represent standard deviations.
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Figure 4: Increased migration of HeLa26 is not a product of wounding
Migration patterns of HeLa26, HeLaPar and HeLaCx26R75Y were tracked with Metamorph
software in sparse cultures
(A) Images for day 0 and 1 are shown along with a selection of individual cell migration tracks
(colored lines) (Video 4).
(B) Tracking confirmed the visual impression that there was no “directionality” to the
movements (i.e. X/Y was ~1 – not shown), but the velocity of cell movement (total
distance travelled in any direction over time) was much higher in HeLa26 cultures (black
bar in left set of data), exceeding that seen in the wound healing assay (right set of data).
(C) Coupling of confluent and sparse cultures was measured by mixing HeLa cells pre-labeled
with a junctional impermeant red dye (DiI) and a junctional permeant green dye (Calcein)
with an excess of unlabeled cells of the same type. After one day of culture, cell coupling
was determined by FACS analysis as the number of cells with green, but no red dye
(bottom right quadrant). This analysis showed minimal dye transfer between HeLa
parental cells in either confluent (2.5%) or sparse (1.2%) conditions, but a substantial level
Journal of Cell Science • Advance article
of cell communication in both confluent (15.6%), and sparse (8.8%) HeLa26cultures.
(A) HeLa WT (left), HeLa43 (center) and HeLa26 (right) scrape wounded monolayers were
stained with N-cadherin (green), β–catenin (red) and DAPI (blue). All showed co-
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Figure 5: N-Cadherin is necessary and sufficient to modify HeLa26 cell migration.
localization of N-cadherin and β–catenin at cell interfaces in the monolayer distal to the
wound (top row), although HeLa26 cells appeared to have lower levels of N-cadherin.
This co-localization was preserved, albeit at lower levels, at the wound edge in HeLaWT
and 43, but HeLa26 showed a reduced levels and cytosolic distributions of N-cadherin,
and nuclear localization of β–catenin. (See Fig. 1S for images of individual clones).
(B) Western blots confirm that HeLa26 cells have reduced levels of N-cadherin compared to
HeLa43 and WT cells
(C) RTqPCR demonstrated that the reduction in N-cadherin in HeLa26 compared to WT and
43 cells was also at the RNA level (* = p<0.0001; n = 3). (also see Fig 2aS for levels in
wounded and unwounded cultures).
(D) Treatment of HeLa43 cells with an siRNA targeted to N-cadherin reduced protein
expression to levels by 50% compared to scrambled siRNA treated control (although still
3 times higher that HeLa26),
(E) Treatment with N-cadherin siRNA, but not a scrambled form, increased the degree of
wound closure seen in 2 days (* = p<0.0001; n = 3) to close to that of HeLa26 cells. (3
(F) Transient overexpression of N-Cadherin in HeLa26 raises the levels of N-cadherin protein
to a level similar to that in HeLa WT cells.
(G) The overexpression of N-cadherin in HeLa26 decreases the percentage of wound closure
over 2 days significantly (* = p<0.005; n = 3), although not to the level of HeLa WT
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independent experiments). (see Fig 2bS for parallel experiments with HeLaWT).
other motility factors.
(A) Western Blot of total (left lanes – multiple bands due to posttranslational modifications)
and cytosolic p120 catenin (i.e. cytosolic – right lanes) shows comparable levels of p120
in all cell lines, but a notable increase in the cytosolic levels in HeLa26 cells only.
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Fig. 6: Gap junction induced loss of N-cadherin results in release of catenins that induce
(B) This correlates with activation of Rac, as indicated by an increase in the ATP bound form
in Western blots (B).
(C) siRNA knock down of Rac in HeLa26 produces a 47% drop in N-cadherin protein levels,
(D) This results in a decrease in the rate of wound closure to levels similar to those of HeLa43.
(* p < 0.005; n = 3)
(E) Immunofluorescence of HeLa26 cells distal to the wound (i.e. in the undisturbed
monolayer) and at the wound edge stained with DAPI (nucleus) and for N-cadherin (green)
and β-catenin (red) show the loss of N-cadherin at the wound edge, and the redistribution
of β-catenin from the membrane to the nucleus (also seen in Fig. 5A). This is also
illustrated in the fluorescent emission profiles (right panels) along the line shown in the
fluorescent image. (See Fig. 3S for levels of β-catenin, and co-localization numbers)
(F) The nuclear location of β-catenin is associated with enhanced RNA levels of uPAR, an
activator of matrix metalloproteinases, in HeLa26. This can be mimicked by transfection
of HeLa parental cells with β-catenin. (* p < 0.005 compared to HeLa43 or WT; n = 3)
(G) Similarly, other reported targets of β-catenin also show selective increases in expression
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in HeLa26 cells, such as Collagen IV. (* p < 0.005 compared to HeLa43 or WT; n = 3)
Figure 7: Model of how GJIC induces enhanced motility
The loss of N-Cadherin at the wound edges in HeLa26 cells causes the cells to be less adhesive
and more prone to separation from the region distal to the wound. This also causes displacement
respectively. Free cytosolic p120ctn activates Rac to Rac-GTP, which increases cell migration by
acting on the actin cytoskeleton and stress fibers. Nuclear redistribution of β-catenin increases the
transcription of migration promoting genes likes uPAR and collagen. The Cx26 permeants that
move between cells to induce the original drop in N-cadherin transcription remain to be identified,
but they are apparently not permeable through Cx43 channels.
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of the N-cadherin binding proteins p120 catenin and β-catenin to the cytosol and nucleus
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The distribution of cAMP in wounded cultures of HeLa parental (A), HeLa26 (B) and HeLa43 (C)
cultures was followed indirectly by immunostaining with phospho-PKA antibodies that recognize
an activated form of the enzyme. The number of cells (Frequency) recorded for each range of
pPKA activation (measured in pixel intensities), as a measure of cAMP levels, were plotted for
cells distal to the wound (i.e. in the undisturbed monolayer - red bars) and at the wound edge
Journal of Cell Science • Advance article
Figure 8: cAMP intercellular transfer does not seem to mediate changes in motility
(yellow bars). In the uncoupled HeLa parental cultures, PKA was clearly activated at the wound
edge (right shift of the yellow bars compared to the red), while in the coupled HeLa26 and 43
cultures, the differences in PKA activity between cells at the wound edge and the monolayer were
eliminated (superimposition of the red and yellow distributions), presumably due to equilibration
of cAMP pools. In HeLa26 cultures, while the mean PKA activity at the wound edge and distally
are the same, distribution at the wound edge were broader, presumably because the reduced
adhesiveness of HeLa26 cells caused them to separate and be less coupled.
Acknowledgements:
This study was supported by Grant CA 48049.from the NCI, GM 55437from NIGMS and startup funding from UTHSCSA. The authors would like to thank Drs. Rongpei Lan and Dr.
Manjeri Venkatachalam, Department of Pathology, UTHSCSA, for designing the stamp
wounding device.
Competing interest statement
Author Contributions:
Srikanth Polusani helped to perform the migration studies, and developed the procedures for
measuring coupling in sparse cultures. He also performed all analyses of signaling pathways, and
helped in the writing of the manuscript, contributing to its intellectual content.
Journal of Cell Science • Advance article
The authors declare no competing financial interests.
Edward Kalmykov developed the video procedures for following migration and quantitatively
measuring rates, including the labeling procedures for heterologous cultures. He also contributed
to some of the signaling analyses and writing of initial drafts of the manuscript, contributing to
its intellectual content.
Anjana Chandresekhar helped to develop and characterize all cell lines used in these studies, and
contributed to the early design of the project.
Shoshana Zucker shared major responsibility for the original conception and design of the
project, and hence shares credit for senior authorship. She also performed initial measurements
using the wound healing assay, and served as the direct supervisor of Ms. Chandrasekhar.
Bruce Nicholson, in conjunction with Dr. Zucker, conceived the original design of the project
and served as mentor and supervisor to Dr. Chandrasekhar, and Drs. Polusani and Kalmykov. He
helped in the design of the signaling studies, and contributed primarily to the intellectual content
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and editing of the manuscript.
ANASTASIADIS, P. Z. & REYNOLDS, A. B. 2000. The p120 catenin family: complex roles in adhesion,
signaling and cancer. J Cell Sci, 113 ( Pt 8), 1319-34.
BEAHM, D. L., OSHIMA, A., GAIETTA, G. M., HAND, G. M., SMOCK, A. E., ZUCKER, S. N., TOLOUE, M. M.,
CHANDRASEKHAR, A., NICHOLSON, B. J. & SOSINSKY, G. E. 2006. Mutation of a conserved
threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominantnegative closed gap junction channel. J Biol Chem, 281, 7994-8009.
BEHRENS, J. 2000. Control of beta-catenin signaling in tumor development. Ann N Y Acad Sci, 910, 21-33;
discussion 33-5.
BEVANS, C. G., KORDEL, M., RHEE, S. K. & HARRIS, A. L. 1998. Isoform composition of connexin channels
determines selectivity among second messengers and uncharged molecules. J Biol Chem, 273,
2808-16.
BLASI, F. & CARMELIET, P. 2002. uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol, 3, 93243.
BUKAUSKAS, F. F. & VERSELIS, V. K. 2004. Gap junction channel gating. Biochim Biophys Acta, 1662, 4260.
CHANDRASEKHAR, A., KALMYKOV, E. A., POLUSANI, S. R., MATHIS, S. A., ZUCKER, S. N. & NICHOLSON, B.
J. 2013. Intercellular redistribution of cAMP underlies selective suppression of cancer cell
growth by connexin26. PLoS One, 8, e82335.
CHURKO, J. M. & LAIRD, D. W. 2013. Gap junction remodeling in skin repair following wounding and
disease. Physiology (Bethesda), 28, 190-8.
COON, M. & HERRERA, R. 2002. Modulation of HeLa cells spreading by the non-receptor tyrosine kinase
ACK-2. J Cell Biochem, 84, 655-65.
COUTINHO, P., QIU, C., FRANK, S., TAMBER, K. & BECKER, D. 2003. Dynamic changes in connexin
expression correlate with key events in the wound healing process. Cell Biol Int, 27, 525-41.
DEFAMIE, N., CHEPIED, A. & MESNIL, M. 2014. Connexins, gap junctions and tissue invasion. FEBS Lett,
588, 1331-8.
DENG, Y., CHEN, Y., REUSS, L. & ALTENBERG, G. A. 2006. Mutations of connexin 26 at position 75 and
dominant deafness: essential role of arginine for the generation of functional gap-junctional
channels. Hear Res, 220, 87-94.
EK-VITORIN, J. F. & BURT, J. M. 2013. Structural basis for the selective permeability of channels made of
communicating junction proteins. Biochim Biophys Acta, 1828, 51-68.
ELIAS, L. A., TURMAINE, M., PARNAVELAS, J. G. & KRIEGSTEIN, A. R. 2010. Connexin 43 mediates the
tangential to radial migratory switch in ventrally derived cortical interneurons. J Neurosci, 30,
7072-7.
ELIAS, L. A., WANG, D. D. & KRIEGSTEIN, A. R. 2007. Gap junction adhesion is necessary for radial
migration in the neocortex. Nature, 448, 901-7.
ETIENNE-MANNEVILLE, S. & HALL, A. 2001. Integrin-mediated activation of Cdc42 controls cell polarity in
migrating astrocytes through PKCzeta. Cell, 106, 489-98.
EZUMI, K., YAMAMOTO, H., MURATA, K., HIGASHIYAMA, M., DAMDINSUREN, B., NAKAMURA, Y., KYO,
N., OKAMI, J., NGAN, C. Y., TAKEMASA, I., IKEDA, M., SEKIMOTO, M., MATSUURA, N., NOJIMA, H.
& MONDEN, M. 2008. Aberrant expression of connexin 26 is associated with lung metastasis of
colorectal cancer. Clin Cancer Res, 14, 677-84.
FRANCIS, R., XU, X., PARK, H., WEI, C. J., CHANG, S., CHATTERJEE, B. & LO, C. 2011. Connexin43
modulates cell polarity and directional cell migration by regulating microtubule dynamics. PLoS
One, 6, e26379.
Journal of Cell Science • Advance article
References:
Journal of Cell Science • Advance article
GIEPMANS, B. N. 2004. Gap junctions and connexin-interacting proteins. Cardiovasc Res, 62, 233-45.
GOLDBERG, G. S., LAMPE, P. D. & NICHOLSON, B. J. 1999. Selective transfer of endogenous metabolites
through gap junctions composed of different connexins. Nat Cell Biol, 1, 457-9.
GOLIGER, J. A. & PAUL, D. L. 1995. Wounding alters epidermal connexin expression and gap junctionmediated intercellular communication. Mol Biol Cell, 6, 1491-501.
GOODENOUGH, D. A. & PAUL, D. L. 2003. Beyond the gap: functions of unpaired connexon channels. Nat
Rev Mol Cell Biol, 4, 285-94.
GRAEBER, S. H. & HULSER, D. F. 1998. Connexin transfection induces invasive properties in HeLa cells.
Exp Cell Res, 243, 142-9.
GROSHEVA, I., SHTUTMAN, M., ELBAUM, M. & BERSHADSKY, A. D. 2001. p120 catenin affects cell
motility via modulation of activity of Rho-family GTPases: a link between cell-cell contact
formation and regulation of cell locomotion. J Cell Sci, 114, 695-707.
HARRIS, A. L. & CONTRERAS, J. E. 2014. Motifs in the permeation pathway of connexin channels mediate
voltage and Ca (2+) sensing. Front Physiol, 5, 113.
HSU, M., ANDL, T., LI, G., MEINKOTH, J. L. & HERLYN, M. 2000. Cadherin repertoire determines partnerspecific gap junctional communication during melanoma progression. J Cell Sci, 113 ( Pt 9), 153542.
HUANG, G. Y., COOPER, E. S., WALDO, K., KIRBY, M. L., GILULA, N. B. & LO, C. W. 1998. Gap junctionmediated cell-cell communication modulates mouse neural crest migration. J Cell Biol, 143,
1725-34.
ICHII, T. & TAKEICHI, M. 2007. p120-catenin regulates microtubule dynamics and cell migration in a
cadherin-independent manner. Genes Cells, 12, 827-39.
JONGEN, W. M., FITZGERALD, D. J., ASAMOTO, M., PICCOLI, C., SLAGA, T. J., GROS, D., TAKEICHI, M. &
YAMASAKI, H. 1991. Regulation of connexin 43-mediated gap junctional intercellular
communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin. J Cell Biol, 114,
545-55.
KAMIBAYASHI, Y., OYAMADA, Y., MORI, M. & OYAMADA, M. 1995. Aberrant expression of gap junction
proteins (connexins) is associated with tumor progression during multistage mouse skin
carcinogenesis in vivo. Carcinogenesis, 16, 1287-97.
KANCZUGA-KODA, L., SULKOWSKI, S., LENCZEWSKI, A., KODA, M., WINCEWICZ, A., BALTAZIAK, M. &
SULKOWSKA, M. 2006. Increased expression of connexins 26 and 43 in lymph node metastases
of breast cancer. J Clin Pathol, 59, 429-33.
KEANE, R. W., MEHTA, P. P., ROSE, B., HONIG, L. S., LOEWENSTEIN, W. R. & RUTISHAUSER, U. 1988.
Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in
neuroectoderm. A study in vitro. J Cell Biol, 106, 1307-19.
KOTINI, M. & MAYOR, R. 2015. Connexins in migration during development and cancer. Dev Biol, 401,
143-51.
KRETZ, M., EUWENS, C., HOMBACH, S., ECKARDT, D., TEUBNER, B., TRAUB, O., WILLECKE, K. & OTT, T.
2003. Altered connexin expression and wound healing in the epidermis of connexin-deficient
mice. J Cell Sci, 116, 3443-52.
LAIRD, D. W. 2010. The gap junction proteome and its relationship to disease. Trends Cell Biol, 20, 92101.
LAMPE, P. D. & LAU, A. F. 2000. Regulation of gap junctions by phosphorylation of connexins. Arch
Biochem Biophys, 384, 205-15.
LAN, R., GENG, H., HWANG, Y., MISHRA, P., SKLOSS, W. L., SPRAGUE, E. A., SAIKUMAR, P. &
VENKATACHALAM, M. 2010. A novel wounding device suitable for quantitative biochemical
analysis of wound healing and regeneration of cultured epithelium. Wound Repair Regen, 18,
159-67.
Journal of Cell Science • Advance article
LIN, J. H., TAKANO, T., COTRINA, M. L., ARCUINO, G., KANG, J., LIU, S., GAO, Q., JIANG, L., LI, F.,
LICHTENBERG-FRATE, H., HAUBRICH, S., WILLECKE, K., GOLDMAN, S. A. & NEDERGAARD, M.
2002. Connexin 43 enhances the adhesivity and mediates the invasion of malignant glioma cells.
J Neurosci, 22, 4302-11.
MCNEIL, P. L., MURPHY, R. F., LANNI, F. & TAYLOR, D. L. 1984. A method for incorporating
macromolecules into adherent cells. J Cell Biol, 98, 1556-64.
MIEKUS, K., CZERNIK, M., SROKA, J., CZYZ, J. & MADEJA, Z. 2005. Contact stimulation of prostate cancer
cell migration: the role of gap junctional coupling and migration stimulated by heterotypic cellto-cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer
cells. Biol Cell, 97, 893-903.
MITRA, S. S., XU, J. & NICHOLSON, B. J. 2012. Coregulation of multiple signaling mechanisms in pp60vSrc-induced closure of Cx43 gap junction channels. J Membr Biol, 245, 495-506.
MUSIL, L. S., CUNNINGHAM, B. A., EDELMAN, G. M. & GOODENOUGH, D. A. 1990. Differential
phosphorylation of the gap junction protein connexin43 in junctional communicationcompetent and -deficient cell lines. J Cell Biol, 111, 2077-88.
NOREN, N. K., LIU, B. P., BURRIDGE, K. & KREFT, B. 2000. p120 catenin regulates the actin cytoskeleton
via Rho family GTPases. J Cell Biol, 150, 567-80.
OGAWA, K., PITCHAKARN, P., SUZUKI, S., CHEWONARIN, T., TANG, M., TAKAHASHI, S., NAIKI-ITO, A.,
SATO, S., ASAMOTO, M. & SHIRAI, T. 2012. Silencing of connexin 43 suppresses invasion,
migration and lung metastasis of rat hepatocellular carcinoma cells. Cancer Sci, 103, 860-7.
POLLMANN, M. A., SHAO, Q., LAIRD, D. W. & SANDIG, M. 2005. Connexin 43 mediated gap junctional
communication enhances breast tumor cell diapedesis in culture. Breast Cancer Res, 7, R522-34.
PON, Y. L., AUERSPERG, N. & WONG, A. S. 2005. Gonadotropins regulate N-cadherin-mediated human
ovarian surface epithelial cell survival at both post-translational and transcriptional levels
through a cyclic AMP/protein kinase A pathway. J Biol Chem, 280, 15438-48.
REYNOLDS, A. B., DANIEL, J., MCCREA, P. D., WHEELOCK, M. J., WU, J. & ZHANG, Z. 1994. Identification of
a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol
Cell Biol, 14, 8333-42.
REYNOLDS, A. B., DANIEL, J. M., MO, Y. Y., WU, J. & ZHANG, Z. 1996. The novel catenin p120cas binds
classical cadherins and induces an unusual morphological phenotype in NIH3T3 fibroblasts. Exp
Cell Res, 225, 328-37.
SAEZ, J. C. & LEYBAERT, L. 2014. Hunting for connexin hemichannels. FEBS Lett, 588, 1205-11.
SNEYD, J., WILKINS, M., STRAHONJA, A. & SANDERSON, M. J. 1998. Calcium waves and oscillations driven
by an intercellular gradient of inositol (1,4,5)-trisphosphate. Biophys Chem, 72, 101-9.
SOLAN, J. L. & LAMPE, P. D. 2014. Specific Cx43 phosphorylation events regulate gap junction turnover in
vivo. FEBS Lett, 588, 1423-9.
SOSINSKY, G. E. & NICHOLSON, B. J. 2005. Structural organization of gap junction channels. Biochim
Biophys Acta, 1711, 99-125.
TATE, A. W., LUNG, T., RADHAKRISHNAN, A., LIM, S. D., LIN, X. & EDLUND, M. 2006. Changes in gap
junctional connexin isoforms during prostate cancer progression. Prostate, 66, 19-31.
VALIENTE, M., CICERI, G., RICO, B. & MARIN, O. 2011. Focal adhesion kinase modulates radial gliadependent neuronal migration through connexin-26. J Neurosci, 31, 11678-91.
WILLECKE, K., EIBERGER, J., DEGEN, J., ECKARDT, D., ROMUALDI, A., GULDENAGEL, M., DEUTSCH, U. &
SOHL, G. 2002. Structural and functional diversity of connexin genes in the mouse and human
genome. Biol Chem, 383, 725-37.
XU, X., LI, W. E., HUANG, G. Y., MEYER, R., CHEN, T., LUO, Y., THOMAS, M. P., RADICE, G. L. & LO, C. W.
2001. Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap
junctions. J Cell Biol, 154, 217-30.
Journal of Cell Science • Advance article
ZHANG, W., MAO, Y-Q., WANG, H., Yin, W-J., Zhu. S-X. & WANG, W-C. 2015. miR-124 suppresses cell
motility and adhesion targeting talin 1 in prostate cancer cells. Cancer Cell International 15, 4958.
ZHU, H., WANG, H., ZHANG, X., HOU, X., CAO, K. & ZOU, J. 2010. Inhibiting N-cadherin-mediated
adhesion affects gap junction communication in isolated rat hearts. Mol Cells, 30, 193-200.
ZONG, L., ZHU, Y., LIANG, R. & ZHAO, H-B. 2015. Gap junction mediated miRNA intercellular transfer and
gene regulation: a novel mechanisms for intercellular genetic communication. Scientific Reports
6, 19884-19892.
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
VIDEO LINKS for Poulisani et al. (JOCES/2105/185017)
Four Videos showing the various migration assays presented in Fig 1 (Video 1), Fig 3
(Videos 2 and 3) and Fig 4 (Video 4) have been uploaded to Figshare, as they are larger than
the 5MB limit for Supplementary files. The links are provided below each caption.
(NOTE: As the movies have multiple frames displayed, you need to download the file to view them,
as the Preview only shows one frame)
VIDEO DESCRIPTIONS:
1. VIDEO 1 – HeLa parental and HeLa26 confluent cultures were subjected to a scrape
wound and allowed to close the wound over 28 hours. The timelapse video shows how
the HeLa parental cells close the wound slowly by progressing into the wounded area as a
cohesive “sheet”, maintaining close cell contact. In contract, the HeLa cells expressing Cx26
move more quickly to fill the wound, frequently breaking contact with the main monolayer
and actively migrating independently, showing much higher rates of filopodial extension
and activity. Quantification of motility rates shown in Fig 1
https://figshare.com/s/bbd9ddb582b1a0f0637c
2. VIDEO 2 – Homotypic (top two panels) or Mixed (bottom panel) confluent cultures of
HeLa26 (green) and HeLa43 (red) were subjected to a scrape wound and followed as
in Video 1, but with longer spacing between frames. Cx26 and Cx43 are known not to form
heterotypic channels, so these two cell lines do not couple. Each cell type was labeled by a
lipohilic dye (DiI (Cx43) and DiO (Cx26), and the color distribution in each cell averaged by
Metamorph software. In the mixed culture (lower panel) the green cells (HeLa26) fill the
wound more rapidly than the red ones (HeLa43), each migrating at rates similar to what
they do in homotypic cultures (shown in panels above). Quantitation shown in Fig 3B
3. VIDEO 3 - Homotypic (top two panels) or Mixed (bottom panel) confluent cultures of
HeLa26 (green) and HeLa32 (red) were subjected to a scrape wound and followed as
in Video 2. Cx26 and Cx32 are known to form heterotypic channels and these cell lines do
couple. Cells were labeled as in Video 2. In this case, in the mixed culture, the green
(HeLa26) and red cells (HeLa32) fill the wound equally, as the HeLa32 cells now show
higher motility than in their homotypic culture, approximating the motility rate of HeLa26
cells. Quantitation shown in Fig. 3B
https://figshare.com/s/0aaa849fd009bec0209f
4. VIDEO 4 – Sparse cultures of HeLa parental (top left), HeLa26 (top right) and HeLa26
R75Y (a mutant that forms hemichannels but not gap junctions – bottom) were
monitored over 15 hours by time lapse video. HeLa26 showed dramatically higher
motility and activity in filopodial extensions than the other HeLa cells. Since the only
property that distinguishes Cx26 and Cx26R75Y is the ability to form intercellular
channels, this demonstrated that despite the sparse cultures, intercellular exchange of
metabolites is required for the enhanced motility. Quantitation of rates on cell movement
are shown in Fig 4.
https://figshare.com/s/addf823e0d98c4759a1f
Journal of Cell Science • Supplementary information
https://figshare.com/s/c05749d9046bdffdc077
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
SUPPLEMENTARY FIGURES - Polusani et al JOCES/2015/185017)
Distal monolayer Wound Edge D19 HeLa43 M HeLa26 11 Fig. 1S: N-cadherin (green), β-catenin (red) and DAPI (blue) staining of
two HeLa43 and HeLa26 clones distal to, and at, the wound edge.
Journal of Cell Science • Supplementary information
K7 J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
Figure 1S: Cadherin and beta-catenin staining of HeLa43 and 26 clones
(linked to Fig 5A)
Cultures of two individual clones of HeLa43 cells (D19 and K7) and two clones of HeLa26 (M
and 11) were wounded and stained for N-cadherin (green), β-catenin (red) and DAPI, to highlight
the nuclei. Images are taken in the unwounded monolayer distal to the wound (left) and at the edge
of the wound (right). The co-localization of N-cadherin and β-catenin that was seen at the
interfaces between cells in all clones examined, was largely maintained at the wound edge in
HeLa43 cells, but was lost in the HeLa26 cells. Here the cadherin was redistributed to the cytosol
(and overall levels appear to decrease), and β-catenin appears in the nucleus (superimposition of
Journal of Cell Science • Supplementary information
red and purple labeling).
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
N-­‐cadherin levels in wounded and unwounded cultures
N-Cadherin mRNA levels
A 26
siRNA:
(-)
Scr
HeLa WT
N-Cad
(-)
HeLa 26
Fig 2S: N-­‐cadherin during wound healing of HeLa Journal of Cell Science • Supplementary information
B J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
Figure 2S: N-cadherin levels and modulation during wound healing
(linked to Figs 5C and E)
(A) N-cadherin mRNA levels, measured by RTqPCR from confluent (clear bars) and stamp
wounded cultures, showed that Cx26, and to a lesser extent, Cx32, induced decreased Ncadherin expression in confluent cultures compared to HeLa WT or 43 cells. This was
much greater in wounded cultures, where N-cadherin message in HeLa26 was barely
detectable. HeLa32 cells also showed a significant drop in N-cadherin in wounded
cultures.
(B) In parallel studies to those shown in Figure 5E, siRNA knockdown of N-cadherin in
HeLaWT cells induced them to significantly increase their rate of wound healing to close
Journal of Cell Science • Supplementary information
to that seen in HeLa26 cells.
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
A
26M
43A
WT
T135A
26M
43A
WT
T135A
B-Catenin
N-Cadherin
Actin
Unwounded
Stamp Wounded
B
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Cx
WT 43 43
clone
D19 K7
location distal monolayer
nucleus-N-cad
nucleus-b-cat
N-cad-b-cat
26
26 26
C
M 11
wound edge
WT 43 43
D19 K7
distal monolayer
26 26 26
C
M
11
wound edge
Fig 3S: β-catenin and N-cadherin in HeLa WT, 43 and 26
Journal of Cell Science • Supplementary information
% Colocaliation
Colocalization of N-cadherin & b-catenin
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
Figure 3S: β-catenin and N-cadherin levels and distribution in unwounded and wounded
cultures (linked to Figs 5A and 6E)
(A) Western blots of β-catenin, N-cadherin and Actin (as loading control) from confluent
(unwounded) and wounded cultures of different HeLa transfectants show reductions of
N-cadherin and β-catenin in HeLa26 unwounded cultures. In wounded cultures, there is a
complete loss of N-cadherin protein in HeLa26, and a partial loss in HeLa cells expressing
the non-functional Cx26T135A mutant and in HeLa32 cells. However, β-catenin remains
present in all cell lines after wounding, although at reduced levels in HeLa26.
(B) The relative localization of N-cadherin and β-catenin immunofluorescent signals with the
nuclear compartment (associated with DAPI staining) and with one another was assessed
using Metamorph software on a Nikon TE 2000 microscope in HeLaWT, HeLa43 clones
(DM19 and K7) and HeLa26 clones (C, M and 11) distal to, and at the edge of the wound,
(see images in Fig. 1S). Generally patterns were similar, except for the HeLa26 clones at
evident, along with a drop in N-cadherin-β-catenin co-localization (in all clones except
HeLa26 M).
Journal of Cell Science • Supplementary information
the wound edge, where an increase in β-catenin (and N-cadherin) nuclear localization was
% wound healed in 2 days
J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
120
Fibronectin
Collagen
Laminin
NoECM
100
80
60
40
20
0
26
32 WT
Fig 4S: Effect of different ECM components on mobility of HeLa Journal of Cell Science • Supplementary information
cells expressing different Cxs. J. Cell Sci. 129: doi:10.1242/jcs.185017: Supplementary information
Figure 4S: Motility of different HeLa cell lines on various extracellular matrices
(linked to Figs 6 F and G)
Since HeLa26 cells express collagen (Fig 6 G) to a greater degree than HeLa WT or Cx32 and 43
transfectants. We examined how each clone migrates (measure as % of wound closure after 48
hours) with or without coating of the plates with extracellular matrix components. HeLa26 shows
no difference in migration rate on any matrix compared to plastic, while HeLa32 and WT both
showed enhanced migration with collagen and fibronectin, with no, or slight inhibitory affects
Journal of Cell Science • Supplementary information
associated with laminin.