For an epithelium to provide a protective barrier, it must maintain homeostatic cell
numbers by matching the number of dividing and dying cells. While compensatory cell
division can be triggered by dying cells
1–3
, how cell death might relieve overcrowding due to proliferation is not known. When
we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional
barrier
4
. To extrude, cells destined to die signal surrounding epithelial cells to contract
an actomyosin ring that squeezes the dying cell out
4–6
. However, it is not clear what drives cell death during normal homeostasis. We show
that overcrowding, due to proliferation and migration, induces extrusion of live cells
to control epithelial cell numbers. Live cell extrusion occurs at sites where highest
crowding occurs in vivo and can be induced by experimentally overcrowding monolayers
in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding
also requires Sphingosine 1-Phosphate (S1P) signalling and ROCK-dependent myosin contraction
but is distinguished by signalling through stretch-activated channels. Moreover, disruption
of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads
to the formation of epithelial cell masses. Our findings reveal that during homeostatic
turnover, growth and division of epithelial cells on a confined substratum causes
overcrowding that leads to their extrusion and consequent death due to loss of survival
factors. These results suggest that live cell extrusion could be a tumour suppressive
mechanism that prevents the accumulation of excess epithelial cells.
To examine how cells are eliminated during homeostasis, we immunostained human colon
tissues, developing zebrafish epidermis, and cultured Madin-Darby Canine Kidney (MDCK)
epithelial cells for active-caspase-3 to identify apoptotic cells and actin and DNA
to highlight cell borders and extrusion
4, 6
. Surprisingly, we found predominantly caspase-negative cells extrude from the surfaces
of colon epithelia (80%, n=46 extruding cells), from zebrafish epidermis (88%, n=160
extruding cells in 3 experiments), and from overgrown monolayers in culture (67%,
n=300 extruding cells in 3 experiments; Fig. 1 a,b,d,e,g,h). By contrast, extrusions
resulting from inducing apoptosis in zebrafish with G418 or cultured monolayers with
ultraviolet-C (UV-C) were almost exclusively caspase-3 positive
6
. These findings suggest that during homeostasis and development, live rather than
dead cells are eliminated by extrusion. Similar live cell extrusions have been observed
during mammary gland involution
7
. Because these non-apoptotic in vivo extrusions looked identical to apoptotic extrusion,
we hypothesized that the extrusion pathway operates in two diverging manners: one
to maintain homeostatic cell numbers in epithelia and the other to remove apoptotic/damaged
cells.
When quantifying extrusion in both adult human colon and developing zebrafish epidermis,
we noticed that extrusions occurred predominantly at the fin edges and colon surfaces,
where cell densities were highest (1.7- and 1.8-fold higher than fin center or crypt
side (Supplementary Fig. 1a&b), respectively, indicated by yellow cells in Fig. 1c,f).
Extrusion zones were also more curved in vivo, yet, because they occurred most frequently
in regions with higher cell density (1.8 fold) compared to proliferative regions in
flat cell culture monolayers (Fig. 1i and Supplementary Fig. 1c), we decided to experimentally
test if overcrowding strain could induce cells to extrude.
To simulate overcrowding observed in vivo, we grew MDCK cells to confluence on a silicone
membrane stretched to 28% of its original length and then released it from stretch
(Fig. 2a). Within 30 minutes after release, the number of cells per 100μm2 increased
1.3-fold, from 112 ±5 to 144 ±4 (Fig. 2b–f). ZO-1 and β-catenin staining confirmed
that tight and adherens junctions were still intact and that the average cell diameter
had decreased by 30 minutes post-crowding (Fig. 2g–i and Supplementary Fig. 2). Moreover,
crowded monolayers maintained adhesion to Cy-5-labeled fibronectin coated membranes
(Fig. 2j). By 6 hours, the number of cells per field equilibrated to pre-release levels
(110 ±10 cells/100μm2, see Fig. 2e–f), indicating that MDCK epithelia eliminate cells
to achieve homeostatic cell numbers.
Immunostaining for actin and active-caspase-3 showed that overcrowding induced predominantly
non-apoptotic extrusion, similar to levels during homeostasis (Fig. 3a). Filming crowded
MDCK monolayers expressing Lifeact-GFP
8
to highlight F-actin or the active-caspase-3 indicator NucView™ confirmed that live
cells are extruded by contracting actin rings (Supplementary Movie 1 and Supplementary
Fig. 4). The numbers of live extruding cells eventually decreased by 6 hours post-overcrowding
concordantly with the decrease in cell densities. The percentage of non-apoptotic
extrusion also correlated to the percentage of crowding (Supplementary Fig. 3).
To determine whether live cell extrusions required the same factors that control apoptotic
cell extrusion, we blocked signals required for extrusion during experimental overcrowding.
We previously identified sphingosine-1-phosphate (S1P) as the signal apoptotic cells
produce to activate the S1P receptor 2 (S1P2), which triggers Rho-mediated contraction
to squeeze the dying cells out
5
. Inhibiting Rho Kinase (ROCK) or S1P signalling dramatically decreased the percentages
of both non-apoptotic and apoptotic cell extrusions (Fig. 3b). Although it was not
possible to score non-apoptotic cells blocked from extrusion, as they look like any
other cells within the monolayer, the significant decreases in percentages of non-apoptotic
extrusion indicated that live cell extrusion, like apoptotic extrusion, requires S1P
signalling through ROCK-mediated actomyosin contraction.
To assess the fate of overcrowding-induced extruded cells, we analysed their viability
using flow cytometry and their ability to survive and proliferate by plating them
on new substrata. After collecting extruded cells from the medium two hours post-crowding,
45.3 ± 2.1% were alive, whereas only 21.3 ± 2.7% were alive after UV-induced apoptotic
extrusion (Fig. 3c and Supplementary Fig. 5a). Overcrowding and homeostatically extruded
cells were able to proliferate into confluent monolayers, whereas those collected
from apoptosis-induced extrusion did not (Fig. 3d). If, however, cells extruded from
homeostatic or post-crowded monolayers were collected after 24 hours, most cells died
instead of proliferating after replating (Supplementary Fig. 5b), suggesting that
extruded cells typically die unless a new substratum is provided. Presumably, during
homeostasis in vivo most extruded cells die from anoikis, apoptosis due to loss of
survival signals from engagement with the underlying matrix
9–11
.
We next investigated what signals trigger live cell extrusion during homeostasis or
overcrowding. We previously found that over-expression of Bcl-2 inhibits both apoptosis
and extrusion in response to apoptotic stimuli (
12
and Fig. 3f), however, Bcl-2 overexpression did not block live cell extrusion during
homeostasis or following overcrowding (Figs. 3e, g). To investigate signals that might
regulate live cell extrusion after overcrowding, we tested two candidates that are
activated by cell stress: the c-terminal Jun Kinase (JNK)
13
and stretch-activated channels
14, 15
. JNK II inhibitor blocked apoptotic extrusion in response to UV-C but not live cell
extrusion following overcrowding or homeostatic cell turnover (Figs. 3e–g). In contrast,
we found that inhibiting stretch-activated ion channels with gadolinium (Gd3+)
16, 17
significantly reduced the percentage of both apoptotic and non-apoptotic extrusion
events following overcrowding or during epithelial homeostasis, yet had no effect
on apoptotic cell extrusion in response to UV (Fig. 3e–g). When cell extrusion is
blocked by Gd3+, the number of cells per 100 μm2 remains high at 2 hours post-crowding
(167 +/−12) compared to control crowded cells (112 +/−5), demonstrating that extrusion
relieves overcrowded cell densities. Thus, while Bcl-2 and JNK control apoptosis-induced
extrusion, stretch-activated signalling controls live cell extrusion during homeostasis
that is induced by overcrowding, presumably upstream of S1P signalling (Fig. 4l).
If stretch-activated signalling controls extrusion-mediated homeostatic cell turnover,
blocking this pathway in vivo should lead to accumulation of excess epithelial cells.
By immunostaining and filming the epidermis of developing zebrafish with Tg(cldnb:lynGFP)
18
, we found that cells proliferate at 37 hours post fertilization (hpf) and migrate
to the fin edges between 48–59 hpf where they extrude at 60 hpf (Fig. 4a, c–e, Supplementary
Fig. 6, and Movie 2). Treating 48 hpf zebrafish with Gd3+ blocked homeostatic cell
extrusion and resulted in epidermal cell mass formation at the fin edges, the number
of which was dependent on Gd3+ concentration (Fig. 4b, f–h, Supplementary Fig. 7 and
Movie 3). Live imaging of Gd3+-treated Tg(cldnb:lynGFP) zebrafish revealed that the
cell masses formed at zones of convergence, where cells failed to extrude (Fig. 4h
and Movie 4).
By knocking down candidate stretch-activated channels in zebrafish with morpholinos,
we found that live cell extrusion requires the channel Piezo1
14
. To prevent developmental defects from early knockdown (Supplemental Fig. 8a&b),
we used a photo-cleavable morpholino (photo-MO) to knockdown Piezo1 at 30 hpf (Supplemental
Fig. 8c&d)
19
. By 60 hpf, Piezo1 morphants, like Gd3+-treated fish could not extrude cells and
developed epidermal masses at the fin edges and other sites of tissue strain (Figs.
4b, i–k and Supplementary Figs. 9&10), indicating that Piezo1 regulates extrusion
to maintain homeostatic epithelial cell numbers in vivo.
As extrusion appears to control cell numbers during homeostasis, we predict that alterations
in extrusion could lead to various epithelial pathologies. Aberrant signalling or
tensions that block extrusion could lead to formation of cell masses, as those produced
in zebrafish when stretch-activated channels are disrupted. Furthermore, these cell
masses may set the stage for tumourigenesis by allowing retention of oncogenic or
defective cells. In support of this, we found increased cell densities and no clearly
identified extrusions in colon polyp sections compared to control sections (Supplementary
Fig. 11). Conversely, hyper-contraction of bronchial epithelia following bronchoconstriction
in asthmatics could lead to excessive extrusion and characteristic epithelial denuding.
The resulting poor barrier could lead to increased inflammation and infection seen
in asthmatics
20–22
. While mathematical models have suggested that mechanical forces could control tissue
homeostasis
23
, our results show that cell division and migration within epithelia cause overcrowding
strain, which induces live cells to extrude and later die. Thus, homeostatic live
cell extrusion provides the previously missing link between proliferation and epithelial
cell death (Fig. 1c,f,i).
Materials and Methods Summary
All cell culture, cell staining, UV irradiation, and imaging were done using MDCK
cells, as previously described
6
. Cells were treated with 10μM SKI-II (Calbiochem), 10μM JTE-013 (Tocris Bioscience),
10 μM SP600125, 10μM Gd3+, (both Sigma-Aldrich), or 1% DMSO as a control. Flow cytometry
was done use a Beckman-Dickinson FACScan after treating cells with 1μg/250μL of propidium
iodide (Sigma-Aldrich). The Huntsman Cancer Institute Tissue Resource and Applications
Core provided human colon sections. Developing wild-type AB zebrafish were treated
with 10mM Gd3+ at 28.5°C until 60hpf, and immunostained according to
6
or filmed with a Nikon spinning disc confocal microscope using Andor software. For
the photo-MO experiments, the translation blocking antisense morpholino was mixed
at 1:1 molar ratio with a 25bp sense photo-morpholino and injected into 1–2 cell stage
wild-type AB or Et(Gal4-VP16)zc1044a;Tg(UAS-E1b:Kaede)s1999t
zebrafish embryos. At 28-32hpf, embryos were exposed to 350nm light for 20 seconds
to release the caging sense-morpholino, then fixed and immunostained at 60 hpf.
Supplementary Methods
Cell Culture and Overcrowding Assays
Madin-Darby Canine Kidney (MDCK II) cells were grown as described
6
. A custom-design Teflon chamber (left) was fabricated to culture cells on flexible
silicone membranes in stretched states (2×2 cm, 0.5 mm thickness)
24, 25
. Prior to cell seeding, 2×2 cm silicone membranes were stretched and coated with
5μg/mL fibronectin (BD Biosciences) at 4°C for 24 hrs. To stretch membranes with our
device, one edge of the silicone membrane was clamped in place, while the other side
was clamped to a movable shaft, which moves through a Teflon chamber with a sealing
gasket. The movable shaft was pulled out to lengths representing 11%, 22% and 28%
strain. 750,000 cells/ml were plated onto the 2×2 cm silicone matrices in a stretched
state in the device or in a culture dish (for non-stretched controls) and grown to
confluence, then released from their stretched states, thereby crowding cells in monolayer.
Epithelial monolayers were fixed and stained or filmed using a Nikon 90i wide-field
fluorescence microscope. Bcl-2 over-expressing cell lines are described in
12
.
Drug and UV Assays
Confluent MDCK monolayers on silicone membranes were pre-treated with SP600125 JNK
II inhibitor at 10, 50, and 100 μM or 10, 50, and 100 μM Gadolinium III Chloride (Gd3+)
for 30 minutes at 37°C. Apoptosis was induced by exposing monolayers to 1,200 μJ/cm2
UV254 irradiation in a UV series II (Spectroline). Cells were fixed and stained from
1–6 hrs post-UV treatment depending on the experiment. The minimal drug concentrations
(10 μM) established in the UV-induced extrusion assay were used in the overcrowding
and homeostatic assays. For testing extrusion following overcrowding, chambers were
pretreated with 10 μM JTE-013 (Tocris Bioscience), 10 μM SKI II (Calbiochem) or 10μM
Y-27632 (Tocris) for 30 minutes prior to overcrowding up to 2 hours post-crowding.
Cell Immunostaining
Fixation and staining of MDCK II cells for actin, DNA, active-caspase 3, ZO-1, and
β-catenin was carried out as previously described in
6
. Fibronectin was labeled with an Amersham Cy5 Ab Labeling Kit (GE Healthcare).
Live Cell Imaging
Standard MDCK cells or Lifact-GFP
8
expressing MDCK cells were grown to confluent monolayers and imaged on a Nikon 90i
wide field fluorescent microscope with the stage kept at 37°C. For the Nucview experiments,
cells were incubated with the activated caspase-3 indicator Nucview at 1:200 for 30
minutes prior to imaging.
Colon Sections
Human colon tissue samples were fixed, imbedded in paraffin, and cut into 10 μm sections
by the Huntsman Cancer Institute Tissue Resource and Applications Core. The sections
were deparaffinised and rehydrated by incubating in Citrus Clearing Solvent (CCS-Richard
Allen Scientific), 100%, 95%, 80%, 70% ethanol, and PBS. Antigens were retrieved by
heating the slides in 10 mM Sodium Citrate at 95°C for 20 minutes, then rinsed 3 times
with PBS, blocked with 5% BSA/0.5% Tween-20 in PBS for 24 hours, and incubated overnight
with active-caspase 3 antibody, rinsed 5 times with PBS, incubated in Alexa488-anti-
rabbit antibody, 1μg/ml Hoechst, and Alexa568-anti-actin antibody for 2 hours, rinsed
3 times in PBS, and mounted in Prolong Gold (Invitrogen).
Microscopy
Images were captured on a CTR6000 microscope (Leica) with a 63x oil lens using a Micromax
charge-coupled device camera (Roper Scientific) or on a Nikon Eclipse TE300 inverted
microscope converted for spinning disc confocal microscopy (Andor Technologies) using
a 20x or 60X plan fluor 0.95 oil lens with an electron-multiplied cooled CCD camera
1000 × 1000, 8 × 8 mm2 driven by the IQ software (Andor Technologies). Image J was
used to stack 8–10 consecutive 1μm confocal sections into Z series, which were then
colour-combined and reconstructed into a 3D image using Metamorph (GE Healthcare).
IP Lab 4.0.8 s and Image-J software were used to analyse percentages of apoptosis
and extrusion. For quantification of extrusion in tissue culture, extruding cells
were manually scored as non-apoptotic extruding, apoptotic extruding, or blocked apoptotic
extrusions based on the presence of an actin ring and caspase-3 staining in 10,000
monolayer cells.
Statistical Analyses
Statistical analysis was done on at least four independent experiments for the overcrowding
control and time analysis. Three separate assays were used for drug treatments and
for FACS analysis. The error bars in all figures are the standard error of the mean
(s.e.m.). All p-values were determined from a two-sided unpaired student’s t-test
using GraphPad Prism software.
Fluorescent Activated Cell Scanning (FACS) and proliferation analysis
Confluent homoeostatic, UV, and overcrowding monolayers were rinsed 3 times with PBS
to remove any previous cells in suspension. The media was replaced and collected at
each denoted analyses time. Media was collected and centrifuged at 3000 rpm in a Damon/IEC
Division Clinical Centrifuge for 1 minute. The cells were resuspended in 250ul of
PBS containing 4μg/ml propidium iodide and analysed by FACS. Samples were analysed
on a Beckman Dickinson FACScan and 50–1000 cells were analysed for propidium iodide
fluorescence at each condition. For proliferation analysis, pelleted cells were instead
replated in a 96 well dish and representative pictures were taken at 24hrs and 5 days
after replating. Results are from three independent experiments.
Zebrafish Care and Maintenance
Adult zebrafish were maintained under standard laboratory conditions, with a regular
light/dark cycle of 14 hours light and 10 hours of darkness. Embryos were collected
and raised in E3 embryo medium at 28.5 °C and staged according to
26
. The Institutional Animal Care and Use Committee (IACUC) at the University of Utah
(Animal Welfare Assurance #10-07017) have approved all procedures performed in this
protocol using the zebrafish, Danio rerio.
Zebrafish Drug Treatments
Zebrafish embryos were dechorionated at 24 hours post-fertilization (hpf). At 32 hpf
approximately 50–100 embryos were transferred to dishes containing E3 embryo medium
with or without (control) 10mM Gd3+ and allowed to develop at 28.5°C until 60 hpf,
when the animals were either imaged live or fixed for immunostaining.
Zebrafish Immunostaining
Developing zebrafish larvae were fixed in 4% formaldehyde for 1–2 hours at room temperature
or overnight at 4°C. Fixed specimens were then permeablised by rinses with PBSTx (0.5%)
and incubated in blocking buffer (1% DMSO, 2mg/mL BSA, 0.5% Triton X-100 and 10% goat
serum in PBS) for 2 hr. Specimens were then incubated overnight in primary antibodies
for phospho-histone H3 (H3P, Abcam, 1:500) or activated caspase-3 (BD Pharmigen, 1:700).
Samples were subsequently washed six times with PBSTx and then incubated in blocking
buffer for 2hr before incubation with the appropriate secondary antibodies or Alexa-phalloidin
(488/568/647) (Invitrogen). After incubation with secondary antibodies or Alexa-phalloidin,
the specimens were rinsed five times with PBSTx, incubated with DAPI (1:1000) for
15 minutes, rinsed once more and then mounted in prolong gold. Depending on the situation,
either whole specimens or only tail fragments (from the yolk extension back) were
mounted for imaging. For quantification of H3P positive cells, the region from the
cloaca back to the edge of the tail fin was quantified.
Zebrafish Imaging
Developing zebrafish were immunostained according to
6
and imaged with either the wide-field fluorescent microscope or a spinning disc confocal,
described above. For live imaging, wild-type or Gd3+ treated developing Tg(cldnb:lynGFP),
a kind gift from T. Piotrowski, zebrafish were anesthetised with 0.02% Tricaine in
E3, mounted in 1% low melt agarose and imaged on a spinning disc confocal using a
20x objective, capturing a z-series every 2 minutes for 3–6 hours. Manual tracking
of individual cells was performed using Metamorph.
Morpholino Antisense Oligonucleotide Knockdown of Piezo1
Developing zebrafish embryos were injected with 2–4ng of a standard translation blocking
antisense morpholino oligonucleotides directed against the 5′UTR of piezo1 (Accesssion#
XM_691263) at the 1–2 cell stage and then allowed to develop at 28.5°C. For the photo-cleavable
morpholino experiments, the translation blocking antisense morpholino (TBMO) was mixed
at a 1:1 molar ratio with a sense-photo morpholino, with a 4bp mismatch around the
photo-linker (see Supplementary Fig. 8c for schematic of strategy) and then injected
into 1–2 cell stage wild-type AB or Et(Gal4VP16)zc1044a;Tg(UAS-E1b:Kaede)s1999t
embryos. At 28-32hpf, the developing embryos were then exposed to 350nm light for
20 seconds using a 10x objective on a Nikon 90i wide-field fluorescent microscope
to activate the morpholino. Some injected embryos were not converted as a control
for “leakiness” of the sense-photo MO. Likewise, some wild-type or Kaede expressing
embryos that were not injected were exposed to 350nm light to ensure photo-conversion
did not cause any adverse effects.
Morpholinos:
FAM38A 5′UTR TBMO: GAGCGACACTTCCACTCACATTCCT
FAM38A UTR pho 4m: AGGAATGTGAaaxttAGTGTCGCTC
Western Blot Analyses of Piezo1
piezo1 TBMO injected animals (n=15–20) were collected at 28hpf and homogenized in
lysis buffer, boiled at 85°C for 5 minutes, spun down and the supernatant collected.
A Bradford assay was performed to assess protein concentration. 10–15μg of protein
was run out on a 3–8% Tris-Acetate gel at 150V for 1 hour. Detection was performed
with an anti-Piezo1 antibody (Proteintech Cat#15939-1-AP) using standard ECL (enhanced
chemiluminescence) methods. HC11, a mammary epithelial cell line, was used as a control
for protein levels in mammalian tissues. Piezo1 proteins levels are presented relative
to the α-tubulin loading control. Quantification of the scanned blot was performed
using Image J.
Zebrafish embryos expressing the photoconvertable protein Kaede in the epidermis
To generate the Et(Gal4VP16;myl7:GFP)zc1044a
enhancer-trap line, a plasmid along with tol2 mRNA was injected into 1-cell stage
developing zebrafish embryos. The potential founders were then crossed to Tg(UAS-E1b:Kaede)s1999t
28 and subsequently identified by Kaede expression. The identified F1 transgenic embryos
were then imaged at 2 and 5 days post-fertilization for identification and characterization
of epithelial specific expression patterns.
Supplementary Material
1
2