Fig 2: E-cadherin is an instructive cue for mitotic spindle orientation.(a) Schematic representations of full-length and T151 truncated E-cadherin (under control of the doxycycline (DOX) repressible promotor). Immunostaining of endogenous LGN and the HA-tagged T151 E-cadherin (-DOX) showing that the plasma membrane localization of LGN is no longer restricted to cell–cell contacts (arrowheads) but also localizes at membranes not in contact with neighbouring cells as well (asterisks). (b) Confocal images of ?-tubulin labelled centrosomes in MDCK T151 cells showing the orientation of the mitotic spindle in metaphase. (c) Quantification of mitotic spindle angle relative to the basal surface in MDCK T151 cells. (d) Localization of LGN in L-cells in the absence or presence of ectopic E-cadherin (E-cad). (e) Confocal images of ?-tubulin labelled centrosomes in L-cells, showing the orientation of the mitotic spindle in metaphase, in the absence or presence of ectopic E-cadherin. (f) Quantification of mitotic spindle angle relative to the basal surface in L-cells in the absence (empty vector) or presence of ectopic E-cadherin. Scale bars, 10 µm. Quantified data were pooled from three independent experiments. Grey bars in dot plots show mean±s.d.; ****P<0.0001 using Mann–Whitney test.

Fig 3: E-cadherin localizes the mitotic LGN/NuMA complex to cell–cell contacts.(a) S-protein agarose pull down of recombinant LGN–TPR and E-cadherin cytoplasmic tail in the presence or absence of NuMA peptide, immunblotted for LGN and E-cadherin, showing the competition of E-cadherin cytosolic tail from LGN–TPR:S-tag by the LGN-binding domain of NuMA. (b) Localization of endogenous NuMA and E-cadherin in interphase and mitotic MDCK cells. (c) TIRF and epifluorescence microscopy of NuMA in MDCK cells plated on micro-patterned alternating stripes of collagen-IV/E-cad:Fc. Cells are outlined in yellow (mitotis) and grey (interphase), for increased clearness stripes are shown as projections in the TIRF image. (d) Quantification of NuMA fluorescence intensity at the plasma membrane bound to stripes coated with different proteins in interphase and mitotic cells. (e) Cytosolic localization of over-expressed GFP-NuMA in interphase MDCK cells, showing its cytosolic accumulation (marked with an asterisk) in cells with high expression. (f) Localization of GFP-NuMA on MDCK cells plated on collagen-IV/E-cad:Fc micro-patterned surfaces visualized by TIRF microscopy. (g) Quantification of NuMA fluorescence intensity at the plasma membrane bound to different stripes in mitotic cells on shRNA-mediated depletion of LGN, disruption of LGN/Gai interactions by overexpression of Ric8, or on expression of siRNAs targeting Gai1, Gai2 and Gai3. Representative images and epifluorescence of NuMA localization in LGN shRNA monolayers are shown in Supplementary Fig. 4a–4c. (h) Model for localization of the LGN/NuMA complex to cell–cell contacts by E-cadherin. By interacting with the cytosolic tail of E-cadherin, LGN is recruited to cell–cell contacts in interphase. After nuclear breakdown during mitotic entry, NuMA is released from the nucleus and competes LGN from the E-cadherin/LGN complex. The locally formed LGN/NuMA complex remains at E-cadherin adhesions through the interaction of LGN with Gai. Scale bars, 10 µm; all quantified data were pooled from three independent experiments; grey bars in dot plots show mean±s.d.; ****P<0.0001; ***P=0.0002, N.S.=not significant, using unpaired student t-test.

Fig 4: An artificial E-cadherin cell–cell junction orients epithelial cell divisions.(a) Design of the E-cadherin:Fc sidewall in which a vertical silicone sidewall is coated with E-cadherin:Fc (E-cad:Fc) and the basal surface is uncoated. (b) Localization of E-cadherin:DsRed (E-cad) at the sidewall-cell junction in MDCK cells. (c) Immunostaining of endogenous LGN in cells associated with an E-cadherin:Fc sidewall, showing that LGN is recruited to the sidewall-cell junction marked with E-cadherin (arrowhead), similar to native cell–cell adhesions (arrow) in the monolayer, whereas LGN is absent from the free membrane (asterisk). (d,e) GFP-tubulin expressing MDCK cells associated with E-cad:Fc (n=20) and Fc control (n=21) sidewalls, with rose diagram quantification of the mitotic spindle angle relative to the sidewall. See Supplementary Fig. 3f for unbinned data. Scale bars, 10 µm. Quantified data were pooled from four independent experiments.

Fig 5: LGN is recruited to cell–cell contacts directly by E-cadherin.(a) Localization of endogenous LGN at cell–cell contacts, marked with E-cadherin (E-cad), in interphase and mitotic MDCK cells. Arrowheads identify cell–cell contacts, and asterisks mark plasma membrane not contacting another cell. (b) TIRF and epifluorescence microscopy imaging of endogenous LGN in MDCK cells plated on surfaces micro-patterned with alternating stripes of collagen-IV/E-cad:Fc or collagen-IV/Fc, with a quantification of LGN staining intensities at the plasma membrane bound to different stripes. Quantified data were pooled from three independent experiments, grey bars show mean±s.d. (c) Surface structures of NuMA in complex with the TPR repeats of LGN20, and of ß-catenin and p120-catenin with E-cadherin3233. For more details of the E-cadherin/p120-catenin and LGN/NuMA-binding interface, see Supplementary Fig. 2. CBD, catenin binding domain; JMD, juxtamembrane domain. (d) GST-pull down of recombinant E-cadherin cytosolic tail (GST-E-cad-cyto) with LGN–TPR, immunoblotted for GST (red) and LGN (green). (e) U2OS cells, with low levels of endogenous E-cadherin, showing recruitment of YFP-tagged LGN–TPR to E-cadherin cytosolic tail (cyto) or JMD targeted to mitochondria by fusion with the mitochondrial localization signal (MLS) of ActA. This recruitment was not observed with CBD or the JMD containing a DEE758-760AAA mutation. EV, empty vector. Scale bars, 10 µm; ****P<0.0001; N.S=not significant, using paired student t-test.