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Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II.

Yashiro H, Loza AJ, Skeath JB, Longmore GD - Mol. Biol. Cell (2014)

Bottom Line: We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin-containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia.This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling.This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin-containing vesicular trafficking during AJ remodeling in live epithelia.

View Article: PubMed Central - PubMed

Affiliation: ICCE Institute, Washington University School of Medicine, St. Louis, MO 63110 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110.

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Production of Rab11-positive REs and AJ rescue does not require MLCK- or Rho1-mediated actin reorganization. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and pMLC (A′′) in AJ region of GFP-marked PECs expressing wild-type MLCK (MLCK). Confocal immunofluorescence localization of DE-cad (red; B, C, C′), pMLC (B′), and Rab11 (C′′) in AJ region of GFP-marked PECs coexpressing Rho1 RNAi and MLCK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat map of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PEC (D) and PECs coexpressing Rho1 RNAi and MLCK (D′). Confocal immunofluorescence localization of DE-cad (red; E, E′) and pCofilin (E′′) in AJ region of GFP-labeled PECs expressing wild-type LIMK. Confocal immunofluorescence localization of DE-cad (red; F, G, G′), pCofilin (F′), and Rab11 (G′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and LIMK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PECs (H) and PECs coexpressing Rho1 RNAi and LIMK (H′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White scale bars (lower right corner), 10 μm.
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Figure 6: Production of Rab11-positive REs and AJ rescue does not require MLCK- or Rho1-mediated actin reorganization. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and pMLC (A′′) in AJ region of GFP-marked PECs expressing wild-type MLCK (MLCK). Confocal immunofluorescence localization of DE-cad (red; B, C, C′), pMLC (B′), and Rab11 (C′′) in AJ region of GFP-marked PECs coexpressing Rho1 RNAi and MLCK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat map of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PEC (D) and PECs coexpressing Rho1 RNAi and MLCK (D′). Confocal immunofluorescence localization of DE-cad (red; E, E′) and pCofilin (E′′) in AJ region of GFP-labeled PECs expressing wild-type LIMK. Confocal immunofluorescence localization of DE-cad (red; F, G, G′), pCofilin (F′), and Rab11 (G′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and LIMK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PECs (H) and PECs coexpressing Rho1 RNAi and LIMK (H′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White scale bars (lower right corner), 10 μm.

Mentions: The kinase MLCK can also phosphorylate MLC to stimulate myosin II activity, and MLCK is not regulated by Rho1-Rok. MLCK overexpression resulted in increased pMLC staining in both wild-type and Rho1-deficient PECs, as expected (Figure 6, A′′ and B′); however, its overexpression in Rho1-depleted clones was unable to restore AJs (Figure 6C′; quantified in Supplemental Table S3) or Rab11 staining (Figure 6C′′; quantified in Figure 6, D and D′, and Supplemental Figure S7C).


Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II.

Yashiro H, Loza AJ, Skeath JB, Longmore GD - Mol. Biol. Cell (2014)

Production of Rab11-positive REs and AJ rescue does not require MLCK- or Rho1-mediated actin reorganization. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and pMLC (A′′) in AJ region of GFP-marked PECs expressing wild-type MLCK (MLCK). Confocal immunofluorescence localization of DE-cad (red; B, C, C′), pMLC (B′), and Rab11 (C′′) in AJ region of GFP-marked PECs coexpressing Rho1 RNAi and MLCK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat map of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PEC (D) and PECs coexpressing Rho1 RNAi and MLCK (D′). Confocal immunofluorescence localization of DE-cad (red; E, E′) and pCofilin (E′′) in AJ region of GFP-labeled PECs expressing wild-type LIMK. Confocal immunofluorescence localization of DE-cad (red; F, G, G′), pCofilin (F′), and Rab11 (G′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and LIMK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PECs (H) and PECs coexpressing Rho1 RNAi and LIMK (H′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White scale bars (lower right corner), 10 μm.
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Figure 6: Production of Rab11-positive REs and AJ rescue does not require MLCK- or Rho1-mediated actin reorganization. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and pMLC (A′′) in AJ region of GFP-marked PECs expressing wild-type MLCK (MLCK). Confocal immunofluorescence localization of DE-cad (red; B, C, C′), pMLC (B′), and Rab11 (C′′) in AJ region of GFP-marked PECs coexpressing Rho1 RNAi and MLCK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat map of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PEC (D) and PECs coexpressing Rho1 RNAi and MLCK (D′). Confocal immunofluorescence localization of DE-cad (red; E, E′) and pCofilin (E′′) in AJ region of GFP-labeled PECs expressing wild-type LIMK. Confocal immunofluorescence localization of DE-cad (red; F, G, G′), pCofilin (F′), and Rab11 (G′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and LIMK. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Yellow pointed arrowheads denote disrupted AJs. Representative heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild-type PECs (H) and PECs coexpressing Rho1 RNAi and LIMK (H′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White scale bars (lower right corner), 10 μm.
Mentions: The kinase MLCK can also phosphorylate MLC to stimulate myosin II activity, and MLCK is not regulated by Rho1-Rok. MLCK overexpression resulted in increased pMLC staining in both wild-type and Rho1-deficient PECs, as expected (Figure 6, A′′ and B′); however, its overexpression in Rho1-depleted clones was unable to restore AJs (Figure 6C′; quantified in Supplemental Table S3) or Rab11 staining (Figure 6C′′; quantified in Figure 6, D and D′, and Supplemental Figure S7C).

Bottom Line: We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin-containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia.This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling.This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin-containing vesicular trafficking during AJ remodeling in live epithelia.

View Article: PubMed Central - PubMed

Affiliation: ICCE Institute, Washington University School of Medicine, St. Louis, MO 63110 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110.

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Related in: MedlinePlus