Epithelial cell-cell contact stimulates β-actin mRNA zipcode-mediated local monomer synthesis. (A) De-convolved epifluorescence images of a single plane of MDCK cells after 3 h in Ca2+ recovery media stained for de novo β-actin monomer synthesis (red) and mature GFP-β-actin (green). Top left: Untreated MDCK cells expressing FL β-actin (FL β-actin). Bottom left: Untreated MDCK cells expressing β-actin with a deleted 3′ UTR (Δ3′ UTR β-actin). Top right: MDCK cells expressing FL β-actin treated with β-actin zipcode antisense oligonucleotides during Ca2+ recovery (FL β-actin + β-actin zipcode AS). Bottom right: MDCK cells expressing FL β-actin treated with β-actin zipcode sense oligonucleotides (FL β-actin + β-actin Sense). Arrows show cell contact sites and arrowheads point to prominent β-actin synthesis sites near cell contact sites. (B) Graph of the average number of β-actin monomer synthesis sites per cell contact. (C) Estimated number of contact site-localized β-actin monomers synthesized during 3 h post-contact derived from the number of observed β-actin monomer synthesis sites for the experiments shown in panel A. (D) Western blots representing the amount of FL β-actin and Δ3′ UTR β-actin being stably expressed in MDCK cells. Scale bars, 10 μm. Error bars represent mean ± SEM based on five cells from three independent experiments. (*) P < 0.001.
