Hammer, E. is mediated by myosin phosphatase via controlled activation and deactivation of myosin II and HDAC6. This regulates the surface density of 51 integrin to modulate fibronectin matrix assembly and governs rates of cell migration and branching morphogenesis. cultures of SMG can be genetically and Grazoprevir pharmacologically manipulated to study morphogenesis in detail40. We first immunostained SMGs to compare the physiological localization of myosin II, which correlates with local contractility41, and acetylated microtubules. Myosin IIA localized to the basal side of the epithelium, but acetylated microtubules localized to the apical side of the epithelial cells, the exact opposite of myosin IIA (Fig. 6a). A similar non-overlapping localization of myosin IIA and acetylated microtubules was observed in HFFs (Supplementary Fig. S5a). Moreover, treatment of SMG with TSA caused outer bud relaxation, partial flattening of the glands, and fewer buds, indicating inhibition Rabbit Polyclonal to B-RAF of branching morphogenesis (Fig. 6b). Although it is not clear which specific cells are causing these changes, these morphological changes were similar to the effects caused by low doses of blebbistatin (data not shown), suggesting that the interplay between actomyosin contraction and microtubule acetylation was phenocopied in intact SMG explants. Notably, similar to the findings in fibroblasts, TSA-induced hyperacetylation of microtubules in SMG increased 51 integrin (Fig. 6c) and FN staining (Fig. 6d) at the junction between epithelium and mesenchyme. In addition, the TSA-treated glands lost the distinctive localization of myosin IIA and acetylated microtubules (Supplementary Fig. S5b). Grazoprevir Open in a separate window Figure 6 Submandibular gland (SMG) explants also reciprocally regulate contractility and microtubule acetylation. (a) Confocal image of E13 mouse SMG explant grown on a filter membrane and immunostained for acetylated microtubules (red), myosin IIA (green), or E-cadherin (blue). In the bottom row, a representative area (hatched box) depicting the junction between the epithelium and mesenchyme is shown. (b) Representative brightfield images of paired mouse SMG explants at E13 treated with 20 M TSA. (c, d) Representative confocal images of paired mouse SMG explants grown on a filter membrane and treated with 20 M TSA and immunostained for 5 integrin (c, green) or FN (d, green) with E-cadherin (red). In the bottom row, a Grazoprevir representative area (hatched box) depicting Grazoprevir the epithelium-mesenchyme junction is shown. (e) Representative brightfield images of mouse SMG explants infected with lentiviruses encoding the indicated proteins. Paired SMGs infected with GFP or GFP-WT-MT, and GFP-HypoAcMT or GFP-HyperAcMT. (f) Maximally projected confocal images of SMG explants expressing indicated proteins and immunostained for E-cadherin (teal), 51 integrins (red, top panels), or FN (red, bottom panels). (g) Hatched boxes in (f) shown enlarged. Intensity of FN (h) or 5 integrin (i) normalized to intensity of E-cadherin and area of the epithelium in pixels. ( 8 glands; n=3) (j) Representative brightfield images of SMG explants co-infected with GFP-tagged HyperAcMT and mApple-tagged phospho-null MLC Grazoprevir (rMLC-AA, negative) or mApple-tagged phospho-mimetic MLC (rMLC-DD, activated). Data are mean SEM.*p 0.05. Bars represent 200 (e or f), 100 (a, b, or j), and 50 (c or d) m. See also Supplementary Figure 5. To verify that the observed morphological changes and the 51 integrin and FN staining on SMG after TSA treatment were due to increased acetylation of microtubules, different tubulin mutants were expressed in SMGs. Lentivirus-mediated expression of these exogenous proteins was extremely low, yet this barely detectable level was effective in mediating characteristic cytoskeletal modifications with no effects on viability of the cultured organs (Supplementary Fig. S5c). Expression of HyperAcMT in SMGs recapitulated the morphological changes seen with TSA (Fig. 6e): the outer bud relaxed and branching morphogenesis was inhibited even at later developmental stages (E12.5 + 3 days). Furthermore, similar to TSA treatment, expression of HyperAcMT.