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Fig. 5.
Colocalization and interdepencies of transcellular array components. (A,B) Imaging of F-actin with moe-GFP (A) and RLC with wtRLC-mCherry (A′) shows colocalization (A′) in both node-and-cable structures with the same relative concentrations in both (B; n=8 regions of interest). (C) Both pRLC and MHC localize to cell cortices in stage 13 explants using both MHC-IIB (red) and mono-phosphorylated RLC (green) (S19-P) antibodies. Levels of both are reduced in RLC morphants (D), and rescued by expression of wtRLC (E), whereas pnRLC expression rescues heavy chain IIB but not pRLC levels (F). At both stage 10 (G,H) and stage 12 (I,J), C-cadherin localization by immunostaining is perturbed in RLC morphant cells (H). Expression of red (K)- and green (L)-labeled C-cadherin in neighboring cells reveals that they colocalize at the membrane (yellow in M), as expected if they have a role in adhesion. Scale bars: 20 µm in A*,G-I and 10 µm in L. |
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Fig. 6.
C-cadherin localization on lamellipodia depends on RLC. Actin is red and C-cadherin or caC-cadherin is green. (A,B) Cadherin forms puncta (arrow) on lamellipodia (A) that resolve into long plaques (arrow), linking actin cables from neighboring cells (B, asterisk). (C) By contrast, lamellipodia on RLC morphant cells maintain a cloud of diffuse C-cadherin signal (arrow) that does not resolve into puncta. (D) Time-lapse microscopy of C-cadherin dynamics in adjacent control cells labeled singly for actin (left) or C-cadherin (right) display adhesions (arrows) that when broken, snap backwards rapidly (asterisk). (E) RLC MO-treated cells (co-injected with a blue dextran) do not make adhesions with neighboring control cells. (F) pnRLC-expressing morphant cells display little protrusive activity or adhesion remodeling (arrow). (G-I) Cells expressing caC-cadherin exhibit longer linear adhesion plaques than C-cadherin-expressing cells (arrows in G,H), which are associated with thicker actin cables (arrow in G′, quantified in I; n=20 widths per condition). Merged images are in G′,H′. Scale bar in A is 20 μm for all images. Error bars represent s.e.m. ***P<0.001. |
