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. Author manuscript; available in PMC: 2016 Dec 22.
Published in final edited form as: Cytoskeleton (Hoboken). 2015 Dec 22;72(12):597–608. doi: 10.1002/cm.21265

Figure 3. Completely mislocalizing β-actin translation blocks adherens junction assembly even with endocytosis inhibition.

Figure 3

MDCK cells with partially mislocalized β-actin translation (expressing Δ3′UTR β-actin mRNA) were pretreated with β-actin mRNA zipcode antisense oligonucleotides and fixed 3 hours after calcium repletion with: (A) DMSO (0.5% v/v) or (B) 80μM dynasore; and immunostained for F-actin (Green), E-cadherin (Red), and Nucleus (Blue). Scale bars = 10μm. Dotted lines indicate cell boundaries and * indicates nucleus of individual cells. ΔZ represents the distance between the apical and basal planes in μm. Note the lamellar overlap in the cells seen in the basal compartment. (C) Mean FCI values. Bars represent Mean ± 95% C.I. One-way ANOVA results for Dynasore with β-actin mRNA zipcode antisense oligonucleotides treatment indicates no significant difference with time in FCI values. Unpaired Student t test indicates no significant variation between Dynasore with antisense oligonucleotides and DMSO with antisense oligonucleotides treatment 3 hours following calcium repletion. (D) Frequency distribution of FCI values for E-cadherin and F-actin following calcium repletion with either Dynasore (1 hour N = 29; 2 hours N = 29; 3 hours N = 28) or DMSO treatment (3 hours N = 22). Bin size = 0.3. Note the proportion of cells with high FCI values remains very low during calcium repletion. (E) Percentage of TRITC-Dextran in the bottom compartment in an in vitro permeability assay following calcium repletion for cells with partially mislocalized β-actin translation (expressing Δ3′UTR β-actin mRNA) and pretreated with β-actin mRNA zipcode antisense oligonucleotides as well as 80μM Dynasore following calcium repletion. The bars are relative to values for cells without any defects in localization of β-actin translation (expressing full length β-actin mRNA). Note barrier integrity fails to recover following calcium repletion upon a more complete mislocalization of β-actin translation with zipcode antisense oligonucleotides.