NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry.
ABSTRACT Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis [1-3]. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion [4-8]. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.
- SourceAvailable from: Clare M Waterman-Storer[Show abstract] [Hide abstract]
ABSTRACT: Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing. Copyright © 2015 Elsevier Ltd. All rights reserved.Current biology : CB. 12/2014;
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ABSTRACT: Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor activated by native collagen. DDRs regulate cell adhesion, migration and various other cell functions. Deletion of the DDR1 gene in mice is associated with a severe decrease in auditory function and substantial structural alterations in a heterogeneous group of cells, including cells containing actin/myosin contractile elements, e.g., outer hair cells (OHCs) (Meyer zum Gottesberge et al. Lab Invest, 88: 27-37, 2008). The non-muscle myosin heavy chain isoform IIA (NM-IIA), encoded by MYH9, is implicated in the regulation of cell spreading, cellular reshaping and movement and cell migration and adhesion. In this study, we identify DDR1 and NM-IIA co-localization in the type III fibrocytes (tension fibrocytes) of the spiral ligament, the OHCs and the stereocilia of both OHCs and inner hair cells. We show for the first time that DDR1 malfunction causes OHC deformation and the separation of the lateral wall, the location of the cellular motor responsible for the electromotile property, explicitly in those regions showing DDR1 and NM-IIA co-localization. On the basis of our results, we propose that DDR1 acts in concert with proteins of the actin/myosin complex to maintain mechanical forces in the inner ear and to stabilize OHC cellular shape for proper auditory signal transduction.Cell and Tissue Research 10/2014; · 3.33 Impact Factor
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ABSTRACT: In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.Nature cell biology. 01/2015;
Current Biology 23, 731–736, April 22, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.03.039
NMII Forms a Contractile Transcellular
Sarcomeric Network to Regulate Apical
Cell Junctions and Tissue Geometry
Seham Ebrahim,1,6Tomoki Fujita,2,6Bryan A. Millis,1
Elliott Kozin,1Xuefei Ma,3Sachiyo Kawamoto,3
Michelle A. Baird,4Michael Davidson,4
Shigenobu Yonemura,5Yasuo Hisa,2Mary Anne Conti,3
Robert S. Adelstein,3Hirofumi Sakaguchi,2,*
and Bechara Kachar1,*
1Laboratory of Cell Structure and Dynamics, National Institute
on Deafness and Other Communication Disorders,
National Institutes of Health, Bethesda, MD 20892, USA
2Department of Otolaryngology-Head and Neck Surgery,
Kyoto Prefectural University of Medicine,
Kyoto 602-8566, Japan
3Laboratory of Molecular Cardiology, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda,
MD 20814, USA
4National High Magnetic Field Laboratory, Florida State
University, Tallahassee, FL 32310, USA
5Electron Microscope Laboratory, RIKEN Center for
Developmental Biology, Kobe 650-0047, Japan
Nonmuscle myosin II (NMII) is thought to be the master inte-
grator of force within epithelial apical junctions, mediating
epithelial tissue morphogenesis and tensional homeostasis
[1–3]. Mutations in NMII are associated with a number of
diseases due to failures in cell-cell adhesion [4–8]. However,
the organization and the precise mechanism by which NMII
generates and responds to tension along the intercellular
junctional line are still not known. We discovered that peri-
odic assemblies of bipolar NMII filaments interlace with
perijunctional actin and a-actinin to form a continuous belt
of muscle-like sarcomeric units (w400–600 nm) around
each epithelial cell. Remarkably, the sarcomeres of adjacent
cells are precisely paired across the junctional line, forming
tion/relaxation of paired sarcomeres concomitantly impacts
changes in apical cell shape and tissue geometry. We show
differential distribution of NMII isoforms across heterotypic
Our results provide a model for how NMII force generation
is effected along the junctional perimeter of each cell and
communicated across neighboring cells in the epithelial
organization. The sarcomeric network also provides a well-
defined target to investigate the multiple roles of NMII in
junctional homeostasis as well as in development and
Results and Discussion
We examine the organization of NMII in the apical junctional
complex (AJC) using the organ of Corti, which is an epithelial
sheet formed by a checkerboard mosaic of sensory (hair cells,
HCs) and nonsensory epithelial cells, flanked medially by a
purely nonsensory epithelium of hexagonally packed inner
sulcus cells (ISCs). We initially sought to investigate the extent
that NMII is involved in regulating the apical perimeter and sur-
face area of the various cell types. To this end, we conducted
a chemical inhibition experiment using the NMII-specific
inhibitor blebbistatin  in explant cultures of the organ of
Corti dissected from P2 mice. Following blebbistatin expo-
sure, the apical surfaces of cells exhibited striking modi-
fications in their perimeter and area when compared with
the control (Figure 1A). These effects were reversed after the
washout of blebbistatin. A morphometric analysis of the
cellular effects of blebbistatin showed a significant (p < 0.01)
increase (3%–30%) in perimeter or junctional length (see Fig-
ure S1A available online), and a corresponding significant
(p < 0.01) increase in apical cell-surface area (Figure S1B).
Upon addition of blebbistatin, the perimeter of HCs also
deviated from circularity, as verified by changes in the
calculated roundness factor (RF, Figure S1C). This loss of
with overall tension reduction at the cell perimeter on addition
of blebbistatin, indicating that the circumferential junctional
actomyosin belt is maintained under tension by NMII. On a
global level, blebbistatin caused a reversible expansion of the
organ of Corti, which was greater along the radial direction as
compared with the longitudinal direction (R/L, Figure S1D).
tional length and apical surface area, as well as concerted
changes in the geometry of the epithelium, on NMII function.
Because our data support a role for NMII in modulating
epithelial apical perimeter we sought to assess the precise
localization of NMII isoforms along the AJC. Immunofluores-
cence of NMIIC and NMIIB showed a remarkable pattern of
distribution as regularly spaced puncta along the perimeter
of each cell. This pattern is clearly observed in both ISCs
(Figure 1B) and in HCs (Figures S1E and S1F). Conversely,
immunoreactivity for NMIIA, a major NMII isoform at stress
fibers and circumferential actin bundles in spreading cells
[10, 11], was barely detectable around the apical perimeter
of these cells (Figure S1G). Measuring the relative fluores-
cence intensity of actin and NMIIB or NMIIC along the
junctional line, we observed an inversely correlated periodic
modulation, with low actin density at the center of the NMII
fluorescence puncta and higher actin density in spaces
between them (Figure 1B, inset), resembling the striated
pattern of myosin and actin in muscle sarcomeres. Strikingly,
fluorescence NMII puncta from adjacent cells consistently
paired in register across the junctional line, appearing collec-
tively as a transcellular network across the epithelial sheet
(Figures 1C and 1D). At tricellular contacts of ISCs, the NMII
fluorescence puncta localized precisely at the corner of each
cell in a regular triangular arrangement (Figure 1C, arrows,
and Figure 1D). The relative fluorescence intensity of NMIIC
puncta was higher at tricellular junctions (5.07 6 0.9, n = 50)
than along bicellular junctions (3.2 6 0.8, n = 50). Because
tricellular junctions experience additional tensions ,
this observation raises the possibility that NMII could be
6These authors contributed equally to this work
*Correspondence: email@example.com (H.S.), firstname.lastname@example.org
distributed in a tension-dependent manner  as part of a
self-regulated tensional homeostasis.
The resemblance of the NMII/actin alternation to that in
muscle sarcomeres prompted us to test for the presence
and distribution of a-actinin, a member of the spectrin family
that crosslinks antiparallel actin filaments in the Z line of
muscle . Immunofluorescence revealed that nonmuscle
a-actinin1 is present along the junctional line in a periodic
pattern alternating with NMII puncta in both ISCs (Figure 1E)
and HCs (Figure S1H). Fluorescence intensity analysis along
the junctional line confirmed the precise alternation of NMII
puncta with regions of high a-actinin1 density, and also the
coincidence of a-actinin1 and actin (Figures 1F, 1G, and S1I).
The average distance between consecutive NMII puncta in
HCs was 436 6 93 nm (median = 479 nm, n = 101), and in
ISCs it was 452 6 65 nm (median = 449 nm, n = 101). The
close-up view in Figure 1G shows the alternation of NMII and
actin/a-actinin1 around the corners of a tricellular junction.
Further confirmation of the periodic pattern of localization of
NMII at the AJC was obtained by exogenously expressing
NMIIC-GFP in organ of Corti explants (Figure S1J).
We tested for orientation of the NMII bipolar filaments
along the junctional line by expressing a double-tagged
NMIIC (with the fluorescence probe mEmerald at the N-termi-
nal myosin head and the fluorescence probe mCherry at the
C-terminal myosin tail) in cultured organ of Corti. Each tail-
specific mCherry fluorescence punctum of the double-tagged
NMIIC appeared as a central red spot flanked on both sides
along the junctional line by green mEmerald head-specific
fluorescence puncta (Figures 2A and 2B). The orientation of
the bipolar filaments parallel to the junctional line was
confirmed using tissue from a transgenic mouse expressing
a GFP-tag at the C-terminal tail of NMIIC that was coimmuno-
labeled with an NMIIC N terminus-specific antibody (Fig-
ure 2C). To assess the length of each array of bipolar NMII
filaments, we measured the distance between the head-
head maxima. The measured length (402 nm 6 53 nm, n =
10) was at the upper end of the reported length distribution
range (w280–400 nm) of NMII bipolar filaments in nonmuscle
cells [15–17] and significantly smaller than muscle myosin II
bipolar filaments, which range from w2 mm in vertebrates
 to w10 mm in invertebrates . Although the lengths
of the bipolar NMII filaments in the AJC were relatively
consistent, some degree of variation was observed in the
separation between bipolar filaments (Figures 2A-2C). These
results are consistent with an arrangement of NMII bipolar
filaments within small regular sarcomeric units, where the
tail regions of NMII are the center points of each sarcomere,
assembled in series to form a belt along the junctional line of
each cell, as illustrated in the model in Figure 2D. The varia-
tion in sarcomere lengths observed is likely due to stochastic
fluctuations in sarcomere contraction/relaxation  or
intrinsic variations in the length of actin and extent of actin
crosslinking by a-actinin .
Labeling the head and tail of NMII additionally provided a
clearer view of the registry between sarcomeres of adjacent
cells across the junctional line (Figure 2C, illustrated in Fig-
ure 2D). NMII puncta at tricellular junctions were also arranged
in a bipolar configuration but formed an angle with the
midpoint tail fluorescence label consistently pointing to the
tricellular corner (Figure 2C, arrows). This suggests that
the midpoint (tail-rich region) of each sarcomere is physically
Figure 1. NMII Regulates Apical Epithelial Geom-
etry and Alternates with Actin and a-Actinin1
along the Apical Junctional Line
(A) Apical surface of mouse organ of Corti explant
cultures with ZO1 (green) and actin (red) labeling,
showing changes in apical geometry of the
epithelia at the cell and tissue level before
(control) and after (blebbistatin) treatment with
blebbistatin and after blebbistatin was washed
out (recovery). OHC, outer hair cells; DC, Deiters’
cells; IPC, inner pillar cells; IHC, inner hair cells;
ISC, inner sulcus cells.
puncta along cell-cell contacts of rat ISCs, with
actin in red. Inset, tracking of red and green fluo-
rescence intensity (FI) along bracketed region
in (B). Arrows in (C) show triangular arrangement
of NMIIC puncta at tricellular contacts.
(D) NMIIC fluorescence puncta in adjacent cells
align precisely across the junctional line (dashed
(E) NMIIC (green) and a-actinin1 (blue) immuno-
fluorescence in ISCs, with actin in red. Arrows
highlight triangular arrangement of NMIIC puncta
at tricellular contacts (arrow).
(F) Magnification of bracket in (E): actin and
a-actinin1 colocalize and alternate with regions
of high NMIIC intensity. Below: corresponding
fluorescence intensity (FI) profile of NMIIC
(green), actin (red), and a-actinin1 (blue).
(G) Magnification of tricellular junction from (E),
showing alternation of NMIIC (green) with actin
(red) and a-actinin1 (blue). Below: corresponding
FI profile of NMIIC (green), actin (red), and a-acti-
Scale bars represent 10 mm in (A) and 3 mm in (B)–
(E). See also Figure S1.
Current Biology Vol 23 No 8
tethered to the tricellular contacts, pinning the tensed sarco-
meric belt to the corners of the cell, whereas the head regions
stay bound to actin filaments, causing the bending of the NMII
sarcomeric units, as illustrated in Figure 2E.
To test for contractility of the NMII sarcomeres, we repeated
the blebbistatin inhibition experiment in rat organ of Corti
cultures and measured changes in the distance between
consecutive NMII immunofluorescence puncta as an indicator
of changes in sarcomere length, as well as changes in junc-
tional length or cell perimeter. Comparing the sarcomere
length before and after 1 hr of 50 mM blebbistatin inhibition,
we observed that the average sarcomere length in control
Figure 2. Sarcomeric Organization and Orientation of Bipolar NMIIC Filaments along the Epithelial AJC
(A) Nonsensory epithelial cell in rat organ of Corti expressing double-tagged NMIIC (N terminus, mEmerald/green; C terminus mCherry/red), with actin in
blue. Inset shows a diagram of diffraction-limited appearance of double-labeled NMII filaments.
(B) Close-up of four bipolar NMIIC filaments from box in (A).
(C) Bipolar NMIIC filaments align end-to-end along the junctional line between ISCs from a NMIIC-GFP mouse, with GFP-tag at NMIIC-tail (green) and anti-
NMIIC-head antibody (red). Arrows point to bipolar arrangement of NMII at tricellular junctions. Inset shows a diagram of the diffraction-limited image of
double-labeled NMII filaments.
across the junctional line.
(E) Model of the arrangement of bipolar NMIIC filaments at a tricellular junction. The ‘‘spring-like’’ symbol represents the putative tether between NMII and the
corner of the cell at tricellular contacts.
washout of blebbistatin (recovery).
(G) Probability distribution (pd) of sarcomere length in ISCs of control (green), blebbistatin (red), and recovery (blue). Dashed lines, measured data; solid
lines, Gaussian fits. Calculated Gaussian widths are: control = 198.4 6 3.7 nm, blebbistatin-treated = 253 6 8.9 nm, and recovery = 200 6 9.3 nm.
(H) ISC apicaljunctionalperimeter incontrol (darkgray), blebbistatin (lightgray), andrecovery(mediumgray) explants. Data representmeans6SD. Asterisk
represents significant to p < 0.01.
Scale bars represent 3 mm. See also Figure S2.
NMII Sarcomeric Network Regulates Apical Junctions
ISCs (461 6 70 nm, n = 418) increased significantly (p < 0.001),
by w24%, with blebbistatin treatment (570 6 97 nm, n = 418)
and was restored (463 6 72 nm, n = 418) after its washout (Fig-
ures 2F and 2G). Similarly, sarcomere length in HCs (424 6
103 nm, n = 467) increased significantly, by w20% (p <
0.001), after blebbistatin treatment (516 6 136 nm, n = 669)
and was reversed (460 6 90 nm, n = 464) after its washout (Fig-
ures S2A and S2B). The blebbistatin-induced changes in
sarcomere length matched the changes in junctional length.
The average junctional length for ISCs (14.3 6 1 mm, n = 100)
increased significantly, by w23% (p < 0.01), after blebbistatin
inhibition (17.6 6 1.1 mm, n = 100) and was restored (14.6 6
0.9 mm, n = 100) after blebbistatin was washed out (Figure 2H).
The average junctional length for HCs (21.3 6 1 mm, n = 100)
increased by w10% (p < 0.01) after blebbistatin inhibition
(23 6 1.1 mm, n = 100) and was restored (21.4 6 1.2 mm, n =
100) after it was washed out (Figure S2C). These results are
Figure 3. Localization of the Sarcomeric Belt
Relative to the Tight and Adherens Junctions of
(A) ISCs of the organ of Corti from a P2 NMIIC-
GFP mouse showing localization of NMIIC-GFP
(green) relative to the tight junction protein clau-
din 9 (red, Cld-9) and adherens junction protein
E-cadherin (blue, E-cad) along the z axis. Optical
sectioning (200 nm/step) is shown top to bottom
from apical toward the basal surface.
(B) Fluorescence intensity (FI) plots of NMIIC
(green), claudin 9 (red), and E-cadherin (blue)
along the z axis.
(C) ISCs from a NMIIC-GFP mouse costained for
actin (red). Optical sectioning (200 nm/step) to-
ward the basal surface reveals the characteristic
NMIIC-GFP fluorescence signal (green), which
peaks at the upper portion of the actin staining.
(D) Fluorescence intensity (FI) of NMIIC (green)
and actin (red) along the z axis.
(E) Diagram illustrating the location of the NMII
sarcomeric belt at the interface of the tight
(claudin 9) and adherens (E-cadherin) junction
components of the AJC.
Scale bars represent 4 mm. See also Figure S3.
‘‘purse-string’’ model [22, 23] for cortical
between sarcomeres across the junc-
tional line was maintained regardless
of contractile state (Figure 2F, arrows),
providing further evidence for the exis-
tence of some form of mechanical
coupling or tight functional coordination
 of sarcomeres across the junctional
line. This is likely an important compo-
nent of a physical network, along which
the force balance interplay between ten-
sions generated at the AJC and cyto-
skeleton of a single cell is transmitted
across the epithelia as a whole.
To examine the precise location of the
NMII sarcomeric belt in relation to the
stratified two-layer (tight and adherens
junction) organization of the AJC, we ac-
quired z stacks of confocal images at
50 nm intervals of the apical junctional
region of organ of Corti tissue from NMIIC-GFP mice, cos-
tained for tight and adherens junction-specific proteins,
claudin 9 and E-cadherin, respectively. Figure 3A shows a
fluorescence signal of claudin 9 along the z axis peaked at the
apical-most region of the AJC, and that of E-cadherin was at
the basal-most side. The fluorescence intensity of NMIIC-
GFP overlapped partially with both claudin 9 and E-cadherin
immunofluorescence (Figure 3A). Of note, the apical-basal
distribution of NMIIC and claudin 9 persisted together at
tricellular junctions (Figure 3A, arrows), deeper in the basal
direction than immunofluorescence at bicellular junctions.
This observation, combined with the knowledge that the
network of tight junctions extends basolaterally when con-
verging at tricellular contacts [25, 26], suggests an interaction
between the tail domain of NMII and tricellular tight junction
components (Figure 2E).
Current Biology Vol 23 No 8
To estimate the relative localization of each tagged protein
along the z axis, we plotted the immunofluorescence intensity
values after correcting for axial (z axis) chromatic aberration
We found that the NMIIC-GFP fluorescence maxima were
w108 6 27 nm (n = 13) below the claudin 9 fluorescence max-
tion of the NMIIC sarcomeres at a midpoint of the AJC, likely
forming a distinct structure that overlaps partially with both
the tight junction and the adherens junction. We also assessed
the localization of NMIIC relative to the total circumferential
actin belt by labeling organ of Corti tissue from a P2 rat with
NMIIC and Alexa Fluor 568-conjugated phalloidin. The z stack
of confocal images obtained (a substack of which is shown in
Figure 3C) showed that the NMIIC fluorescence distribution
does not encompass all the junctional actin but is consistent
with an association with a subset of the circumferential actin
belt (Figure 3D). A model proposing the localization of the
NMII sarcomeric belt within the AJC at the interface of tight
and adherens junctions is presented in Figure 3E.
Double-labeling experiments showed colocalization of
NMIIB and NMIIC along homomeric junctions (e.g., between
ISCs in Figure 4A), suggesting some degree of functional
redundancy. Conversely, NMIIB and NMIIC sarcomeres
consistently distributed asymmetrically across the junctional
line of heteromeric junctions (e.g., between HCs and SCs in
Figure 4C). Because of their differential kinetic properties
, the asymmetric distribution of NMII isoforms may
contribute to the generation of distinct spatiotemporal
distributions of forces across the junctional line and help in
sculpting different cell morphologies within the epithelia.
It is known that mutations in individual NMII isoforms are
linked to the onset and progression of a number of human dis-
eases [4, 5], including hearing loss [4, 6, 7]. To our knowledge,
compensation between NMII isoforms has not been reported,
Figure 4. Differential Distribution of NMII Isoforms and Evidence for Compensation in Absence of NMIIC
(A) Apical surface of ISCs from a P2 NMIIC-GFP mouse immunolabeled with anti-NMIIB antibody. Both NMIIB (red) and NMIIC (green) colocalize and
distribute symmetrically across homomeric junctions between ISCs.
(B) Fluorescence intensity (FI) along bracketed area in (A); NMIIB/NMIIC ratio varies across NMII puncta (asterisk).
(C) The distribution of the NMIIB (red) and NMIIC (green) is asymmetric across heteromeric hair cell (HC)/supporting cell (SC) junctions.
(D and E) Plots of the fluorescence intensity along the HC and SC perimeters within the bracketed region in (C).
(F) ISCs from P6 NMIIC+/+ and NMIIC2/2 mice stained with NMIIC-, NMIIB-, and NMIIA-specific antibodies (green).
(G and H) Quantification of NMIIA, NMIIB, and NMIIC immunofluorescence intensity at cell-cell contacts in NMIIC+/+ (G) and NMIIC2/2 (H). Data are rep-
resented as mean fluorescence intensity 6 SD.
(I) Apical surface of epithelium from the small intestine of a P2NMIIC-GFP mouse showing the periodicdistribution of NMIIC-GFP along the apical perimeter
of enterocytes. Arrows highlight the precise triangular arrangement at tricellular junctions. Inset is a close-up view of the bracketed region, showing that
puncta from adjacent cells line up in register across the junctional line.
(J) A side view of an isolated enterocyte, showing the characteristic actin-based (red) microvilli projecting from the apical surface with the periodic NMIIC
(green) puncta along the apical junctional region.
(K) Close-up view of the periodic NMIIC puncta from the bracketed region in (J).
(L) Probability distribution (pd) of NMIIC sarcomere length in small intestine (red), large intestine (blue), and stomach (green).
Scale bars represent 2.5 mm. See also Figure S4.
NMII Sarcomeric Network Regulates Apical Junctions
However, NMIIC2/2 mice develop normally to at least three
months of age , and the immunofluorescence of NMIIA
and NMIIB in the AJC was significantly increased in these
mice compared to wild-type (Figures 4F–4H), suggesting po-
tential compensatory effects between NMII isoforms.
To determine whether the sarcomeric organization of NMII
is present in other epithelial tissues, we examined the pattern
of distribution of NMIIC in intestinal enterocytes (Figures 4I–
4K) and stomach epithelial cells (data not shown) of the
NMIIC-GFP mouse. The average distance between NMII
puncta was 398 6 58 nm (n = 542) in small intestine, 410 6
66 nm (n = 539) in large intestine, and 414 6 64 nm (n = 488)
in stomach epithelial cells (Figure 4L), indicating a consistent
w400–500 nm range across various epithelial tissues.
In this study, we uncover the likely ubiquitous sarcomeric
organization of NMII within the epithelial AJC. The localization
of the sarcomeric belt at the interfaceof the tightand adherens
junctions provides a well-defined target to investigate the
multiple roles of NMII in tensional homeostasis. Knowledge
pertaining to the differential localization of NMII isoforms can
in the AJC of various tissues during development and disease,
making our findings relevant across biomedical disciplines.
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.
We thank Juan Pablo Inda, Agnieszka Rzadzinska, Felipe Salles, Alex
Rivero, Jian Mao, and Yuhai Dai for their participation in early experiments
and Ethan Tyler for the artwork. This work was supported by the Intramural
Programs of the National Institute on Deafness and Other Communication
Disorders and the National Heart, Lung, and Blood Institute, National
Institutes of Health, and by Ministry of Education, Culture, Sports, Science
and Technology/Japan Society for the Promotion of Science KAKENHI
Grant number 23592491. All rodent procedures were carried out in accor-
dance with NIDCD and NHLBI Animal Care and Use Committee guidelines.
Received: February 4, 2013
Revised: March 12, 2013
Accepted: March 14, 2013
Published: April 4, 2013
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Current Biology Vol 23 No 8