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. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. 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.

Figure 1. NMII regulates apical epithelial geometry and alternates with actin and α-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). (B and C) Localization of NMIIC (green) in periodic puncta along cell-cell contacts of rat ISCs with actin in red. Inset, tracking of red and green fluorescence 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 line). (E) NMIIC (green) and α-actinin1 (blue) immunofluorescence in ISCs, with actin in red. Arrows highlight triangular arrangement of NMIIC puncta at tricellular contacts (arrows). (F) Magnification of bracket in E: Actin and α-actinin1 co-localize, and alternate with regions of high NMIIC intensity. Below: corresponding fluorescence intensity (FI) profile of NMIIC (green), actin (red) and α-actinin1 (blue). (G) Magnification of tricellular junction from E showing alternation of NMIIC (green) with actin (red) and α-actinin1 (blue). Below: Corresponding FI profile of NMIIC (green), actin (red), α-actinin1 (blue). Scale bars: A= 10 μm; B – E= 3 μm. See also Figure S1.

Figure 2. Sarcomeric organization and orientation of bipolar NMIIC filaments along the epithelial AJC

(A) Non-sensory epithelial cell in rat organ of Corti expressing double-tagged NMIIC (N-terminus, mEmerald/green, C-terminus mCherry/red), actin in blue. Inset, 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 NMIIC-GFP mouse. GFP-tag at NMIIC-tail (green), anti-NMIIC-head antibody (red). Arrows point to bipolar arrangement of NMII at tricellular junctions. Inset, diagram of the diffraction limited image of double-labeled NMII filaments. (D) Illustration of the relationship of the sarcomeric belt of an epithelial cell and that of six neighboring cells, showing the paring of individual NMII sarcomeres 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. (F) NMIIC sarcomere length along apical-junctions of ISCs in rat organ of Corti cultures before (control) and after treatment with blebbistatin (blebbistatin), and after 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 ± 3.7 nm, blebbistatin treated = 253 ± 8.9 nm, and recovery = 200 ± 9.3 nm. (H) ISC apical junctional-perimeter in control (dark gray), blebbistatin (light gray), and recovery (mid-gray) explants. Data represent means ± SD. (*) = Significant to p<0.01. Scale bars = 3 μm. See also Figure S2.

Figure 3. Localization of the sarcomeric-belt relative to the tight and adherens junctions of the AJC

(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 claudin 9 (red, Cld-9) and adherens junction protein E-cadherin (blue, E-cad) along the z-axis. Optical sectioning (200 nm/step) 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 an NMIIC-GFP mouse co-stained for actin (red). Optical sectioning (200 nm/step) toward 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 = 4 μm. See also Figure S3.

Figure 4. Differential distribution of NMII isoforms and evidence for compensation in absence of NMIIC

(A) Apical surface of ISCs from P2 NMIIC-GFP mouse immunolabeled with anti-NMIIB antibody. Both NMIIB (red) and NMIIC (green) co-localize 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 panel C. (F) ISCs from P6 NMIIC+/+ and NMIIC−/− 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 NMIIC−/− (H). Data are represented as mean fluorescence intensity ± SD. (I) Apical surface of epithelium from the small intestine of a P2 NMIIC-GFP mouse showing the periodic distribution of NMIIC-GFP along the apical perimeter of enterocytes. Arrows highlight the precise triangular arrangement at tricellular junctions. Inset-Close up view of 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 bracket region in J. (L) Probability distribution (pd) of NMIIC sarcomere length in small intestine (red), large intestine (blue) and stomach (green). Scale bars= 2.5 μm. See also Figure S4.