Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier

Abstract

Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier.

Figure 1. Alix^−/− mice develop bilateral hydrocephalus.

(a) Brains dissected from Alix^−/− and WT mice (n=4) at different ages show overt size difference. (b) Quantification of the ratio between brain volume and body weight (BW) demonstrate that Alix^−/− brains (n=4) from 1-month-old mice are smaller than WT brains from mice of the same age (n=4). (c) MRI of brains from WT (left) and Alix^−/− (right) mice obtained starting at postnatal day 8 until 44 weeks of age. (d) Quantification of the volume of cerebellum, cortex, ventricles and hippocampus 1-month-old mice WT (n=4) and Alix^−/− mice (n=4). Data are shown as mean values±s.d. (e) 3D reconstruction of whole-mount scanning electron microscopy (SEM) images of the CP from the lateral ventricles.

Figure 2. Abnormal cell architecture and AJC in the Alix^−/− CP.

(a) transmission electron microscopy (TEM) revealed the misalignment of the epithelial cells, their irregular shape and size and the prominent blebbing of their microvilli (n=2), Scale bar, 2 μm. (b) SEM of the Alix^−/− CP apical surface showed extensive blebbing (yellow arrows) and large gaps between cells (light blue arrows) (n=2), Scale bar, 2 μm. (c) High-magnification TEM abnormally distended AJC in Alix^−/− CP (n=3), Scale bar, 100 nm. (d) Immunostaining of whole-mount preparations for β-catenin (green), and γ–tubulin (red) demonstrated the abnormal cell shape and mislocalization of the basal bodies in the Alix^−/− CP (n=2). Scale bar, 10 μm.

Figure 3. Motile cilia of lateral wall in Alix^−/− brain show defective orientation.

(a) 3D reconstruction of whole-mount scanning electron microscopy (SEM) images of WT (n=3) and Alix^−/− (n=3) lateral wall. Inset: the typical 9+2 axoneme structure is maintained in Alix^−/− cilia. Scale bar, 100 μm. (b) Transmission electron microscopy (TEM) of the basal bodies in WT and Alix^−/− ependymal cells (n=2). Arrows indicate orientation of the basal bodies. Scale bar, 500 nm.

Figure 4. Alix^−/− epithelia display apoptotic cell extrusion.

(a) 3D confocal reconstruction of WT and Alix^−/− whole-mount CP showing tunnel⁺ and Myosin IIa⁺ cells being extruded from epithelial cell layer of Alix^−/− CP (n=3). (b) SEM images of cluster of cells extruded from Alix^−/− tracheal epithelial cells. Scale bar, 5 μm.

Figure 5. Loss of Alix compromises the epithelial barrier of the CP.

(a) Combination of brain fluorescence and bioluminescence images obtained 24 h after intraperitoneal injection of the dye Evans blue (n=3). (b) 3D confocal analysis of WT and Alix^−/− whole-mount CP shows an increase intracellular flux of FITC-dextran into the Alix^−/− CP compared with the WT; yellow arrowheads indicate points of colocalization between FITC dextran (green) and ZO-1 (red) along the basolateral membrane of the Alix^−/− CP (n=2).

Figure 6. Alix maintains the integrity of actomyosin and segregates with actomyosin together with tight junction proteins.

(a) Confocal images of whole-mount WT and Alix^−/− CP immunostained with phalloidin. Scale bar, 100 μm. (b) Purified actomyosin from WT and Alix^−/− brains immunostained with phalloidin. Scale bar, 10 μm. (c) Brain actomyosin ATPase activities, measured in the presence of Ca²⁺, Mg²⁺ and K⁺. Data are the mean±s.d. of three different actomyosin preparations. (d) Purified actomyosin from WT and Alix^−/− brains analysed by western blots for the presence of Alix and main components of actomyosin, as well as cell junctions and polarity markers. (e) Western blot quantification of actomyosin components. Data shown represent the mean±s.d. of three independent experiments. *P<0.05 by Student's t-test. (f) Immunoprecipitations followed by immunoblottings of subcellular fractions (P2) from WT and Alix^−/− brains.

Figure 7. Alix^−/− CP shows defects in epithelial cell polarity.

(a) Maximum intensity projections (MIP) of confocal microscopy images of whole-mount WT and Alix^−/− CP (n=3) immunostained for ZO-1 (green) and JAM-1 (red). (b) WT and Alix^−/− tracheal epithelial cell cultures immunostained for ZO-1 (green) and Par3 (red). The dotted lines in the XY images indicate the corresponding XZ sections. Data are representative of at least three independent experiments. Scale bar, 10 μm.

Figure 8. Alix is involved in TJ reassembly.

(a,b) Z310 CP cell line transduced with mock or Alix shRNA lentiviral constructs. Cells were treated with 2 mM EGTA or 10 μM cytochalasin D (CD) and allowed to recover. Scale bar, 10 μm.

Figure 9. Model of the mode of action of Alix at the apically restricted actomyosin–tight junction (TJ) complex in the CP.

In normal CP cells, Alix by interacting with F-actin, Par 3 and ZO-1 secures the proper assembly and positioning of an actomyosin–TJ complex at the apical sides of adjacent epithelial cells that defines a spatial membrane domain essential for the maintenance of epithelial cell polarity and barrier. Alix ablation in the CP affects the formation/maintenance of the actomyosin–TJ polarity complex with consequent loss of epithelial polarity and barrier, and progressive hydrocephalus.