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First published online 26 September 2006
doi: 10.1242/jcs.03170

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PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells

¹ Instituto Gulbenkian de Ciência, Ap. 14, Oeiras, Portugal
² Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal

View larger version (81K): [in a new window]   Fig. 1. The PAR polarity complex is localized at the apical membrane of NEP cells. (A) Deduced protein sequence of cPAR3, depicting the aPKC, KIF3A and TIAM1 binding domains (aPKC-BD; KIF3A-BD; TIAM1-BD). (B) Endogenous cPAR3 localizes apically in NEP cells (a). cPAR3 localizes basally to cPAR6 (b,c) and aPKC (d). cPAR6 colocalizes with aPKC (e). (C) cPAR3 colocalizes with N-Cadherin, Afadin and ß-Catenin at the apical tip of NEP cells (a-c). cPAR6 and aPKC are located apically relative to N-Cadherin (d,e). (D) cPAR3 is localized basally relative to both cPALS1 and cCRB3 (a,b). cPAR6 colocalizes apically with cCRB3 (c). (E) LGL1 localizes basolaterally and is absent from the apical cPAR3 accumulation domain (a). (F) Schematic representation of the localization of polarity proteins in NEP cells. L, lumen of the NT. Bars in (Ba,b,d,e;C;Db,c;E), 2 µm; bar in (Bc), 3 µm; bar in (Da), 1 µm.

View larger version (44K): [in a new window]   Fig. 2. cPAR3 colocalizes with cNumb at intercellular junctions. cNumb accumulates at the apical membrane (arrowhead in A) and at cytoplasmic vesicles (arrowheads in B). (C-C'') cPAR3 colocalizes with cNumb at the apical membrane of NEP cells. (D) cPAR3 is co-immunoprecipitated with cNumb in NEP protein lysates. CRMP2 is used as control. Conversely, cNumb co-immunoprecipitates with cPAR3 (E). (F) cNumb accumulates at the apical membrane together with ß-Catenin and N-Cadherin (G). L, lumen of the NT. Magnification in (A), 20x. Bar in (B), 6 µm; bars in (C-C''), 3 µm; bars in (F,G), 2 µm.

View larger version (86K): [in a new window]   Fig. 3. Ectopic cPAR3 accumulates in punctate clusters at the lateral membrane of NEP cells. (A) 12 hours post-electroporation, cPAR3::GFP accumulates in clusters along the plasma membrane of NEP cells (arrows) and also apically (arrowhead). (B) A single electroporated cell with multiple clusters of cPAR::GFP accumulation along the plasma membrane. (C) At 24 hours, cells with high levels of cPAR3 accumulate at the basal region of the NT (arrow) and ectopic mitotic cells (red, pHistH3-positive) are also present in this region (arrowheads in D). (E) At 48 hours, cPAR3 accumulates in rosette-like structures at the basal part of the NE (square), with punctate clusters in other cells (arrow). (F,F') Detail of a rosette-like group of cells [outlined by the dotted line; enlarged from square in (D)], with cPAR3 accumulating at the center (arrow). (G) The cPAR3-N::GFP mutant still localizes apically (arrowhead), but does not form punctate clusters. L, lumen of the NT. Magnification in (A,D), 40x; in (C,E,G), 63x. Bars in (B) and (F,F'), 10 µm.

View larger version (59K): [in a new window]   Fig. 4. cPAR3 overexpression affects positioning of dividing cells within the NE. (A) Normal NEP cells, expressing a membrane-tethered GFP, move their nucleus apically and always divide near the luminal surface (red and white arrowheads). (B) Illustrative example (n=12 cells) of a GFP-gpi NEP cell overexpressing cPAR3 dividing away from the apical surface (red arrow). Bar in (A), 10 µm; (B), 20 µm.

View larger version (85K): [in a new window]   Fig. 5. Ectopic cPAR3 is sufficient to mislocalize polarity and junction-associated proteins in NEP cells. At the electroporated side of the NT, N-Cadherin accumulates ectopically (white bracket in A,A'), colocalizing with cPAR3::GFP punctates (arrowheads in B,B'). The same applies to ß-Catenin (arrowheads in C-D') and aPKC (arrowheads in E-F'). Triple-labelling of cPAR3, cPAR6 and aPKC shows colocalization at ectopic clusters (G,G'). Members of the CRB complex also mislocalize to cPAR3 punctates (arrowheads in H-K'). (L) LGL1 is excluded from the ectopic sites of cPAR3 accumulation at the lateral membrane (arrowheads). b, basal side of the NE. Magnification in (A,A',C,C',E,E',H,H',I,I'), 40x. Bars in (B,B',G,G'), 2 µm; bars in (D,D',F,F',J,J',K,K'), 10 µm; bar in (L), 1 µm.

View larger version (37K): [in a new window]   Fig. 6. PAR complex formation and cNumb subcellular distribution are affected by the presence of excess cPAR3. (A) Western blot shows that overexpression (OE) produces an 80x increase in of cPAR3 levels. (B) Endogenous cPAR6 and aPKC levels are unaffected when cPAR3 is overexpressed. (C,D) Overexpression of cPAR3 recruits more aPKC molecules to the PAR complex. (E) cPAR6/aPKC complex formation is increased in the presence of excess cPAR3. (F) cLGL1 and cNumb protein levels are unaffected when cPAR3 is overexpressed. (G-G'') At 24 hours post-electroporation, cNumb accumulates at the apical region (arrowhead) and in ectopic punctates (arrow). (H,H') Higher magnification of square in (G'') shows that cNumb colocalizes with ectopic cPAR3 punctates (arrowheads), with cNumb vesicles accumulating at the cell periphery (upper half of the image). In the bottom half of the image, showing non-electroporated cells, cNumb-positive vesicles are uniformly distributed in the cytoplasm. Magnification in (G-G''), 20x. Bars in (H,H'), 6 µm.

View larger version (95K): [in a new window]   Fig. 7. Characterization of rosette-like structures in the NE of cPAR3-overexpressing embryos. (A,A') Cycling cells (BrdU+) are present outside the VZ (bars). (B-B'') cPAR3 is localized in a central ring inside the rosettes, together with cPAR6/aPKC. (C,C') Rosette cells (dotted line) contain centrosomes close to the central lumen (dotted circle). (D,D') ß-Catenin (arrowhead) and N-Cadherin (arrowhead in E,E') localize with cPAR3::GFP at the center of rosettes. (F,G) cPAR3 overexpression (left side of the NT) causes an expansion of the cNotch1 and ches5-1 expression domains (white brackets). (H) Ectopic cDelta1 expressing cells can be detected in the basal region of the cPAR3-electroporated NT (white brackets). (I,I') Rosette cells express ches5-1. (J,J') TUJ1- and HU-positive neurons (K,K') accumulate outside rosettes. Magnification in (A,A'), 40x; magnification in (F-H), 20x. Bars in (B-B''), 4 µm; bars in (C-D',J,J'), 10 µm; bars in (I,I'), 5 µm; bars in (K,K'), 20 µm.

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