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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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AJ defect resulting from Rho1 loss is partially restored by Rab11 overexpression. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red) in Rho172F (Rho1 LoF) MARCM PEC clones in the AJ region marked by GFP (A, A′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled Rho172F MARCM PEC clones coexpressing Rab5 RNAi (B, B′). Confocal immunofluorescence localization of DE-cad (red; C, C′, D, D′) and Rab11 (C′′, D′′) in GFP-labeled Rho1 RNAi PEC clones coexpressing Rab11Q70L (constitutively active Rab11; C, C′′) and Rab11wt (wild-type Rab11; D, D′′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled PECs coexpressing Rho1 RNAi and Rab14Q97L (constitutively active Rab14; E, E′), Rho172F MARCM PEC clones coexpressing Sec5 (F, F′), and Sec8 (G, G′). All images were compiled as a sum of multiple confocal slices within the region where AJs were present. Yellow arrowheads indicate disrupted (A, E–G) and rescued (B–D) AJs. White scale bars (lower right corner), 10 μm.
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Figure 3: AJ defect resulting from Rho1 loss is partially restored by Rab11 overexpression. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red) in Rho172F (Rho1 LoF) MARCM PEC clones in the AJ region marked by GFP (A, A′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled Rho172F MARCM PEC clones coexpressing Rab5 RNAi (B, B′). Confocal immunofluorescence localization of DE-cad (red; C, C′, D, D′) and Rab11 (C′′, D′′) in GFP-labeled Rho1 RNAi PEC clones coexpressing Rab11Q70L (constitutively active Rab11; C, C′′) and Rab11wt (wild-type Rab11; D, D′′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled PECs coexpressing Rho1 RNAi and Rab14Q97L (constitutively active Rab14; E, E′), Rho172F MARCM PEC clones coexpressing Sec5 (F, F′), and Sec8 (G, G′). All images were compiled as a sum of multiple confocal slices within the region where AJs were present. Yellow arrowheads indicate disrupted (A, E–G) and rescued (B–D) AJs. White scale bars (lower right corner), 10 μm.

Mentions: To determine whether Rho1 affected Rab11 staining through regulating the formation of Rab11-positive recycling endosomes and thus AJ remodeling, we used the Drosophila pupal eye epithelium to test for genetic interaction between this Rho1 phenotype and a limited number of candidate genes known to positively or negatively regulate intracellular vesicle formation and trafficking (see Supplemental Table S1). We scored the extent to which they restored AJs disrupted by Rho1 loss. All PEC clones were depleted of Rho1 by either UAS-Rho1 RNAi (Rho1 RNAi) or the loss-of-function allele Rho172F (Rho1 LoF), both of which resulted in comparable disruption of AJs between adjacent mutant cells (Figure 3, A and A′, and Supplemental Figure S1, A and A′; quantified in Supplemental Table S1). In all experiments, the extent of rescue of AJs between two adjacent Rho1-deficient cells was quantified by an AJ index (Warner and Longmore, 2009b; see Materials and Methods).


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)

AJ defect resulting from Rho1 loss is partially restored by Rab11 overexpression. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red) in Rho172F (Rho1 LoF) MARCM PEC clones in the AJ region marked by GFP (A, A′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled Rho172F MARCM PEC clones coexpressing Rab5 RNAi (B, B′). Confocal immunofluorescence localization of DE-cad (red; C, C′, D, D′) and Rab11 (C′′, D′′) in GFP-labeled Rho1 RNAi PEC clones coexpressing Rab11Q70L (constitutively active Rab11; C, C′′) and Rab11wt (wild-type Rab11; D, D′′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled PECs coexpressing Rho1 RNAi and Rab14Q97L (constitutively active Rab14; E, E′), Rho172F MARCM PEC clones coexpressing Sec5 (F, F′), and Sec8 (G, G′). All images were compiled as a sum of multiple confocal slices within the region where AJs were present. Yellow arrowheads indicate disrupted (A, E–G) and rescued (B–D) AJs. White scale bars (lower right corner), 10 μm.
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Related In: Results  -  Collection

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Figure 3: AJ defect resulting from Rho1 loss is partially restored by Rab11 overexpression. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red) in Rho172F (Rho1 LoF) MARCM PEC clones in the AJ region marked by GFP (A, A′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled Rho172F MARCM PEC clones coexpressing Rab5 RNAi (B, B′). Confocal immunofluorescence localization of DE-cad (red; C, C′, D, D′) and Rab11 (C′′, D′′) in GFP-labeled Rho1 RNAi PEC clones coexpressing Rab11Q70L (constitutively active Rab11; C, C′′) and Rab11wt (wild-type Rab11; D, D′′). Confocal immunofluorescence localization of DE-cad (red) in GFP-labeled PECs coexpressing Rho1 RNAi and Rab14Q97L (constitutively active Rab14; E, E′), Rho172F MARCM PEC clones coexpressing Sec5 (F, F′), and Sec8 (G, G′). All images were compiled as a sum of multiple confocal slices within the region where AJs were present. Yellow arrowheads indicate disrupted (A, E–G) and rescued (B–D) AJs. White scale bars (lower right corner), 10 μm.
Mentions: To determine whether Rho1 affected Rab11 staining through regulating the formation of Rab11-positive recycling endosomes and thus AJ remodeling, we used the Drosophila pupal eye epithelium to test for genetic interaction between this Rho1 phenotype and a limited number of candidate genes known to positively or negatively regulate intracellular vesicle formation and trafficking (see Supplemental Table S1). We scored the extent to which they restored AJs disrupted by Rho1 loss. All PEC clones were depleted of Rho1 by either UAS-Rho1 RNAi (Rho1 RNAi) or the loss-of-function allele Rho172F (Rho1 LoF), both of which resulted in comparable disruption of AJs between adjacent mutant cells (Figure 3, A and A′, and Supplemental Figure S1, A and A′; quantified in Supplemental Table S1). In all experiments, the extent of rescue of AJs between two adjacent Rho1-deficient cells was quantified by an AJ index (Warner and Longmore, 2009b; see Materials and Methods).

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