Fig 2: Simplified schemes showing the organization of the junctional complexes of polarized epithelial cells in vertebrates.The sub-apical complex includes a structure recently described126 as well the tight junction (TJ) and region ‘below’ this typically described adherens junction (AJ), which includes both Afadin and cadherin complexes. The smaller cadherin punctate junctions along the lateral contacts are not explicitly indicated. Typical non-transmembrane components (for example, p120ctn and ß-catenin) are often used as markers and these are indicative of well-studied components. The Afadin/nectin compartment in vertebrates is often spatially segregated from cadherin as described in the text.

Fig 3: Afadin regulates expression of thermogenic genes in vitro. (a) Two stable Mllt4 shRNA-expressing preadipocyte cell-lines (sh#1 and sh#2) and a GFP shRNA-expressing control preadipocyte cell line (shGFP) were established and knockdown of Afadin confirmed at the protein level. Representative blots are shown. (b+c) Quantification of Bodipy-staining in differentiated cells. RT-qPCR analysis at day 0 and 9 of differentiation of (d) Fabp4 mRNA, (e) Ppar?2 mRNA, (f) Adipoq mRNA and (g) Mllt4 mRNA. Mature adipocytes were stimulated with vehicle or 5 µM CL 316,243 (CL) for 4 h. RT-qPCR analysis at day 0 and day 9 of differentiation with or without CL stimulation of (h) Cidea mRNA, (i) Ppargc1a mRNA, (j) Ucp1 mRNA and (k) Prdm16 mRNA. (l) Immunoblotting from mature adipocytes exposed to vehicle or CL 316,243 for 4 h. N = 3–4. Data are presented as means + SEM, *p < 0.05 vs. shGFP, #p < 0.05 vs. Day 0, $p < 0.05 vs. Day 9.

Fig 4: Two-step model of division orientation.Model of OCD in the embryonic epidermis. During stratification, LGN (green) is recruited to the apical cortex in ~50% of mitoses, promoting perpendicular divisions. For OCDs with perpendicular and planar anaphase orientations, the division angle is fixed at anaphase onset, exhibiting minimal change in radial orientation during telophase. Importantly, the activity of LGN and its binding partners is imprecise, frequently resulting in oblique orientations at anaphase. In these cases, the apical daughter either retains or loses basement membrane contact following cytokinesis (red or blue nuclei, respectively). If contact is maintained, the apical daughter will reorient into a planar position. In contrast, if contact is lost, the apical daughter further stacks above its basal partner. Upon loss of a-E-catenin, vinculin, or afadin, telophase reorientation in either direction fails, resulting in persistent oblique divisions. In comparison, LGN loss reduces perpendicular anaphase orientations, while oblique divisions are properly corrected in a contact dependent manner. Afdn loss on an Gpsm2 mutant background restores oblique divisions and largely rescues the Gpsm2 differentiation defect.

Fig 5: Afadin and contractibility dependent localization of PAK4 to cell–cell junctions.a MDCK cells were treated with non-targeting (NT), Afadin or PAK4 siRNA for 72 h as indicated. Following fixation in methanol, the cells were immuno-stained for PAK4 and Afadin (Mab) as indicated. Confocal images were taken at ×100 magnification. The red-dotted line indicates the boundary of clonal knockdown cells. Repeated in three independent experiments. b Scatter plot showing relative protein levels derived from confocal immuno-fluorescent images. The junctional fluorescence signal was calculated for multiple 10 × 50 pixel junctional regions (cf. red bar, see 'Methods') and displayed as a ratio relative to the NT control (20 cells over 2 independent replicates). Data from two independent experiments were combined with bars indicating standard deviation from the mean analysed using a two-tailed, unpaired t-test (p < 0.0001). Scale bars: 20 µm. c, d MDCK cells were grown to 80% confluence on uncoated glass coverslips, rinsed in calcium-free PBS and incubated in 5 mM EGTA, serum-free DME for 45 min, until more then 50% cells showed rounding. Media containing 5% serum (1.8 mM calcium) was added for 45 min to allow reattachment, before addition of inhibitor/DMSO for 4 h. Cells were then fixed in methanol and immuno-stained for PAK4 or Afadin antibodies and images were taken with an Olympus Fluoview confocal microscope with ×100 oil objective. White arrows indicate enrichment at tricellular junctions. The bicellular junctional fluorescence signal was calculated as above (cf. region with red bar) and tricellular junctional fluorescence signal was calculated for a standard 15 × 15 pixel circle (red) after removal of local background (non-junction) signal. Lower panel shows scatter plots as indicated (20 cells over 2 independent replicates) from two independent experiments. Bars indicate standard deviation from the mean and analysed using an ordinary one-way ANOVA test (****p < 0.0001). Scale bars: 10 µm. Source data are provided as a Source Data file.