Article Title: Telophase correction refines division orientation in stratified epithelia
Figure Lengend Snippet: Afadin is essential for telophase correction and basal contact maintenance (A) Cumulative frequency distribution of telophase division angles from E16.5 epidermis. Afdn knockdown (red) results in random telophase orientation, while uninfected RFP-negative (grey) and littermate (black) controls display a bimodal distribution; n indicates cells analyzed from 3-6 embryos. (B-E) Live imaging of E16.5 Afdn 2711 explants. (B) An obliquely-oriented Afdn 2711 RFP+ cell fails to reorient, while losing basal contact (open arrowhead). (C) Cumulative frequency distributions of division orientation from E16.5 live imaging of Afdn 2711 RFP+ and wild-type littermates; n indicates observed divisions from 3 embryos imaged in 2 separate sessions. (D) Division orientation at anaphase onset (ϕ) and one hour later (θ) for Afdn 2711 RFP+ and RFP-negative cells, plotted from data in (C) . RFP-negative controls correct into a bimodal distribution, while RFP+ cells reorient randomly. (E) Afdn fl / fl primary keratinocytes mosaically infected with Cre-RFP (red) after 8 h 1.5 mM Ca 2+ shift, stained for E-cadherin (green), afadin (red), and phalloidin (grey). Junctions between two uninfected cells (WT:WT) show linear morphology with consistent E-cadherin (green), afadin (red) and phalloidin (grey) labeling. In contrast, junctions between two infected cells are punctate, with less junction-associated phalloidin. (F) Quantification of E-cad continuity along junction length, as in Fig. 4J . (G) Quantification of fluorescence intensity of actin (phalloidin) measured by orthogonal linescans. Phalloidin is decentralized in KO:KO junctions (red) compared to WT:WT (black;. n indicates junctions evaluated. (H) Primary keratinocytes derived from Afdn fl / fl K14-Cre+ embryos ( Afdn cKO ) and Cre-negative littermates after 8 h Ca 2+ shift and stained for α18 (red), α-E-catenin (green), and vinculin (grey). (I) Quantification of vinculin:total α-catenin and (J) α18:total α-catenin fluorescent ratios; n indicates junctions analyzed. (K) Cumulative frequency distribution of E16.5 telophase division angles in Afdn fl / fl , Afdn cKO , and Afdn cKO + Vcl 3466 H2B-RFP epidermis. Vinculin knockdown does not exacerbate Afdn knockout phenotype. Scale bars, 10μm (B) , 20μm (E,H) . P values determined by Kolmogorov-Smirnov test (A,C,K) , student’s unpaired t-test (F,I,J) . * P
Article Snippet: For knockdown screening, primary keratinocytes were seeded at a density of ~150,000 cells per well into 6-well plates and grown to ~80% confluency in E-Low calcium medium and infected with an MOI of ~1.
Techniques: Imaging, Infection, Staining, Labeling, Fluorescence, Derivative Assay, Knock-Out