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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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Regulators of myosin II downstream of Rho1 affect AJ remodeling and Rab11 staining. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in AJ region of GFP-labeled PECs expressing Rok RNAi. Confocal immunofluorescence localization of DE-cad (red; B, B′, C), pMLC (B′′), and Rab11 (C′) in AJ region of GFP-labeled PECs expressing the catalytic domain of Rok (Rok-CAT). White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of GFP-labeled Rho172F (Rho1 LoF) MARCM clones coexpressing Rok-CAT. Heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild type (E) and Rho172F MARCM clones coexpressing Rok-CAT (E′). Confocal immunofluorescence localization of DE-cad (red; F, F′, G), pMLC (F′′), and Rab11 (G′) in AJ region of PECs coexpressing Rho1 RNAi and wild-type Zip (Zip). Representative heat maps of Rab11 immunofluorescence in merged confocal slices in AJ region of wild-type PECs (H) and PECs coexpressing Rho1 RNAi and Zip (H′). Confocal immunofluorescence localization of DE-cad (red; I, I′) and pMLC (I′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and MBS RNAi. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Pointed yellow arrowheads denote restored AJs. Confocal immunofluorescence localization of YFP-Rab5 (green; J, J′), Rab11 (red; J, J′′), and pMLC (blue; J, J′′′) in wild-type PECs ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5). Yellow arrowheads denote vesicles that colocalize YFP-Rab5, Rab11, and pMLC (magnified in inset), quantified as percentage Rab5- and Rab11-positive vesicles that also localize RLC-GFP. 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 5: Regulators of myosin II downstream of Rho1 affect AJ remodeling and Rab11 staining. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in AJ region of GFP-labeled PECs expressing Rok RNAi. Confocal immunofluorescence localization of DE-cad (red; B, B′, C), pMLC (B′′), and Rab11 (C′) in AJ region of GFP-labeled PECs expressing the catalytic domain of Rok (Rok-CAT). White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of GFP-labeled Rho172F (Rho1 LoF) MARCM clones coexpressing Rok-CAT. Heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild type (E) and Rho172F MARCM clones coexpressing Rok-CAT (E′). Confocal immunofluorescence localization of DE-cad (red; F, F′, G), pMLC (F′′), and Rab11 (G′) in AJ region of PECs coexpressing Rho1 RNAi and wild-type Zip (Zip). Representative heat maps of Rab11 immunofluorescence in merged confocal slices in AJ region of wild-type PECs (H) and PECs coexpressing Rho1 RNAi and Zip (H′). Confocal immunofluorescence localization of DE-cad (red; I, I′) and pMLC (I′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and MBS RNAi. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Pointed yellow arrowheads denote restored AJs. Confocal immunofluorescence localization of YFP-Rab5 (green; J, J′), Rab11 (red; J, J′′), and pMLC (blue; J, J′′′) in wild-type PECs ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5). Yellow arrowheads denote vesicles that colocalize YFP-Rab5, Rab11, and pMLC (magnified in inset), quantified as percentage Rab5- and Rab11-positive vesicles that also localize RLC-GFP. 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 altered staining pattern of Rab11 in the AJ region of Rho1 LoF clones was not due to increased apical size of Rho1 LoF clones, as depleting Rok, a major downstream effector of Rho1 that mediates Rho's effects upon actomyosin contractility, also resulted in PECs with increased apical cell size (Supplemental Table S2; Warner and Longmore, 2009a), yet the Rab11 staining pattern was unchanged from that for wild-type cells (Figure 5A′′); nor was it simply the result of disrupting AJs, as clonal loss of DE-cadherin through the homozygous clonal expression of its LoF allele ShgR69 did not reduce Rab11 staining (Supplemental Figure S3, G–G′′). In embryos lacking one genomic copy of Rho1, the level of Rab11 protein was reduced by 25% compared with wild-type embryos (Figure 2G), suggesting that the altered Rab11 staining pattern at the AJ region of Rho1-deficient PECs may, in part, be due to decreased Rab11 protein level. The lethal effects of Rho172F homozygotes and ubiquitously targeted Rho1 RNAi precluded analysis of complete lack of Rho1 upon Rab11 protein levels in vivo. In further controls, the staining pattern of early endosomes (Rab5) and Golgi (Lva and dGM130) were unaffected by Rho1 loss (Figure 2, F–F′′, and Supplemental Figure S4, D and E). Although Rab11 was sufficient to rescue the AJ defect between Rho1-deficient PECs (see later discussion), we were unable to determine whether Rab11 alone was necessary for AJ maintenance due to lethality of Rab11 depletion, even when the caspase inhibitor p35 was concurrently expressed (Supplemental Figure S3, A–A′′). We also tested whether removing a genomic copy of Rab11, through the use of the strongest allele Rab11EP3017, in a heterozygous Rho172F background resulted in AJ disruptions. AJs remained intact in the sensitized background, in which one genomic copy of Rho1 and Rab11 remained (Supplemental Figure S3, J–J′′′). In controls, heterozygous expressions of Rho172F and Rab11EP3017 individually in the whole animal were viable, and pupal eye PECs did not exhibit any AJ defect (Supplemental Figure S3, J′ and J′′).


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)

Regulators of myosin II downstream of Rho1 affect AJ remodeling and Rab11 staining. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in AJ region of GFP-labeled PECs expressing Rok RNAi. Confocal immunofluorescence localization of DE-cad (red; B, B′, C), pMLC (B′′), and Rab11 (C′) in AJ region of GFP-labeled PECs expressing the catalytic domain of Rok (Rok-CAT). White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of GFP-labeled Rho172F (Rho1 LoF) MARCM clones coexpressing Rok-CAT. Heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild type (E) and Rho172F MARCM clones coexpressing Rok-CAT (E′). Confocal immunofluorescence localization of DE-cad (red; F, F′, G), pMLC (F′′), and Rab11 (G′) in AJ region of PECs coexpressing Rho1 RNAi and wild-type Zip (Zip). Representative heat maps of Rab11 immunofluorescence in merged confocal slices in AJ region of wild-type PECs (H) and PECs coexpressing Rho1 RNAi and Zip (H′). Confocal immunofluorescence localization of DE-cad (red; I, I′) and pMLC (I′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and MBS RNAi. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Pointed yellow arrowheads denote restored AJs. Confocal immunofluorescence localization of YFP-Rab5 (green; J, J′), Rab11 (red; J, J′′), and pMLC (blue; J, J′′′) in wild-type PECs ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5). Yellow arrowheads denote vesicles that colocalize YFP-Rab5, Rab11, and pMLC (magnified in inset), quantified as percentage Rab5- and Rab11-positive vesicles that also localize RLC-GFP. 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 5: Regulators of myosin II downstream of Rho1 affect AJ remodeling and Rab11 staining. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in AJ region of GFP-labeled PECs expressing Rok RNAi. Confocal immunofluorescence localization of DE-cad (red; B, B′, C), pMLC (B′′), and Rab11 (C′) in AJ region of GFP-labeled PECs expressing the catalytic domain of Rok (Rok-CAT). White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of GFP-labeled Rho172F (Rho1 LoF) MARCM clones coexpressing Rok-CAT. Heat maps of Rab11 immunofluorescence in merged confocal slices through the AJ region in wild type (E) and Rho172F MARCM clones coexpressing Rok-CAT (E′). Confocal immunofluorescence localization of DE-cad (red; F, F′, G), pMLC (F′′), and Rab11 (G′) in AJ region of PECs coexpressing Rho1 RNAi and wild-type Zip (Zip). Representative heat maps of Rab11 immunofluorescence in merged confocal slices in AJ region of wild-type PECs (H) and PECs coexpressing Rho1 RNAi and Zip (H′). Confocal immunofluorescence localization of DE-cad (red; I, I′) and pMLC (I′′) in AJ region of GFP-labeled PECs coexpressing Rho1 RNAi and MBS RNAi. White arrowheads and asterisks denote wild-type PECs, and yellow arrowheads and asterisks denote mutant PECs. Pointed yellow arrowheads denote restored AJs. Confocal immunofluorescence localization of YFP-Rab5 (green; J, J′), Rab11 (red; J, J′′), and pMLC (blue; J, J′′′) in wild-type PECs ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5). Yellow arrowheads denote vesicles that colocalize YFP-Rab5, Rab11, and pMLC (magnified in inset), quantified as percentage Rab5- and Rab11-positive vesicles that also localize RLC-GFP. 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 altered staining pattern of Rab11 in the AJ region of Rho1 LoF clones was not due to increased apical size of Rho1 LoF clones, as depleting Rok, a major downstream effector of Rho1 that mediates Rho's effects upon actomyosin contractility, also resulted in PECs with increased apical cell size (Supplemental Table S2; Warner and Longmore, 2009a), yet the Rab11 staining pattern was unchanged from that for wild-type cells (Figure 5A′′); nor was it simply the result of disrupting AJs, as clonal loss of DE-cadherin through the homozygous clonal expression of its LoF allele ShgR69 did not reduce Rab11 staining (Supplemental Figure S3, G–G′′). In embryos lacking one genomic copy of Rho1, the level of Rab11 protein was reduced by 25% compared with wild-type embryos (Figure 2G), suggesting that the altered Rab11 staining pattern at the AJ region of Rho1-deficient PECs may, in part, be due to decreased Rab11 protein level. The lethal effects of Rho172F homozygotes and ubiquitously targeted Rho1 RNAi precluded analysis of complete lack of Rho1 upon Rab11 protein levels in vivo. In further controls, the staining pattern of early endosomes (Rab5) and Golgi (Lva and dGM130) were unaffected by Rho1 loss (Figure 2, F–F′′, and Supplemental Figure S4, D and E). Although Rab11 was sufficient to rescue the AJ defect between Rho1-deficient PECs (see later discussion), we were unable to determine whether Rab11 alone was necessary for AJ maintenance due to lethality of Rab11 depletion, even when the caspase inhibitor p35 was concurrently expressed (Supplemental Figure S3, A–A′′). We also tested whether removing a genomic copy of Rab11, through the use of the strongest allele Rab11EP3017, in a heterozygous Rho172F background resulted in AJ disruptions. AJs remained intact in the sensitized background, in which one genomic copy of Rho1 and Rab11 remained (Supplemental Figure S3, J–J′′′). In controls, heterozygous expressions of Rho172F and Rab11EP3017 individually in the whole animal were viable, and pupal eye PECs did not exhibit any AJ defect (Supplemental Figure S3, J′ and J′′).

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