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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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Rho1 function precedes Rab11 recruitment to recycling endosomes by Nuf. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in the AJ region of GFP-labeled MARCM Rho172F (Rho1 LoF) PEC clones coexpressing Nuf. Arrowheads indicate rescued AJs. White asterisk denotes wild-type PEC, and yellow asterisk denotes mutant PEC. Representative heat maps of Rab11 immunofluorescence in merged confocal slices of AJ region in wild type (B) and Rho172F MARCM clones coexpressing Nuf (B′). Confocal immunofluorescence localization of Nuf (red) in GFP-labeled Rho172F MARCM clones (C, C′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Representative heat maps of Nuf immunofluorescence in merged confocal slices through the AJ region in wild type (D) and Rho172F MARCM clones (D′). Confocal immunofluorescence localization of Rab11 (green; E, E′, F, F′) and Nuf (red; E, E′′, F, F′′) in AJ region of wild-type PECs (GMR-Gal4; E, E′′) and PECs ubiquitously depleted of Rho1 (GMR-Rho1 RNAi; F, F′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region. White scale bars (lower right corner), 10 μm. (G) Quantitation of percentage vesicles that localize Nuf alone (pink) or those that colocalize Nuf and Rab11 (yellow). (H) Quantitation of percentage vesicles that localize Rab11 alone (green) or those that colocalize Rab11 and Nuf (yellow). The p values were calculated comparing values in GMR-Rho1 RNAi clones (right) to control GMR-Gal4 (left) using an unpaired, two-sided Student's t test; n.s., p > 0.05.
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Figure 4: Rho1 function precedes Rab11 recruitment to recycling endosomes by Nuf. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in the AJ region of GFP-labeled MARCM Rho172F (Rho1 LoF) PEC clones coexpressing Nuf. Arrowheads indicate rescued AJs. White asterisk denotes wild-type PEC, and yellow asterisk denotes mutant PEC. Representative heat maps of Rab11 immunofluorescence in merged confocal slices of AJ region in wild type (B) and Rho172F MARCM clones coexpressing Nuf (B′). Confocal immunofluorescence localization of Nuf (red) in GFP-labeled Rho172F MARCM clones (C, C′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Representative heat maps of Nuf immunofluorescence in merged confocal slices through the AJ region in wild type (D) and Rho172F MARCM clones (D′). Confocal immunofluorescence localization of Rab11 (green; E, E′, F, F′) and Nuf (red; E, E′′, F, F′′) in AJ region of wild-type PECs (GMR-Gal4; E, E′′) and PECs ubiquitously depleted of Rho1 (GMR-Rho1 RNAi; F, F′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region. White scale bars (lower right corner), 10 μm. (G) Quantitation of percentage vesicles that localize Nuf alone (pink) or those that colocalize Nuf and Rab11 (yellow). (H) Quantitation of percentage vesicles that localize Rab11 alone (green) or those that colocalize Rab11 and Nuf (yellow). The p values were calculated comparing values in GMR-Rho1 RNAi clones (right) to control GMR-Gal4 (left) using an unpaired, two-sided Student's t test; n.s., p > 0.05.

Mentions: Rab11 recruitment to REs is regulated by a family of Rab11-interacting proteins (Rab11-FIPs). In mammals there are two classes of Rab11-FIPs with redundant functions, whereas in Drosophila only two genes have been identified: dRip11, a class I Rab11-FIP, and Nuclear Fallout (Nuf), a class II Rab11-FIP. DRip11 and Nuf are sufficient to increase Rab11 localization and promote RE formation (Shaye et al., 2008; Gault et al., 2012). Therefore we asked whether Rho1 affected the ability of either Drosophila FIP to promote recruitment of Rab11 to vesicles and thereby restore Rab11-positive RE distribution in Rho1-deficient clones. Overexpression of dRip11 restored Rab11 staining pattern in PEC clones devoid of Rho1 (Supplemental Figure S5A′′; quantified in Supplemental Figure S5, B, B′, and C) but was not sufficient to restore AJ organization (Supplemental Figure S5, A and A′; quantified in Supplemental Table S3). On the other hand, Nuf overexpression (Supplemental Figure S5, D and D′) restored both Rab11 staining (Figure 4A′′; quantified in Figure 4, B and B′, and Supplemental Figure S5C) and partially rescued AJs between cells lacking Rho1 (Figure 4, A and A′; quantified in Supplemental Table S3).


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)

Rho1 function precedes Rab11 recruitment to recycling endosomes by Nuf. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in the AJ region of GFP-labeled MARCM Rho172F (Rho1 LoF) PEC clones coexpressing Nuf. Arrowheads indicate rescued AJs. White asterisk denotes wild-type PEC, and yellow asterisk denotes mutant PEC. Representative heat maps of Rab11 immunofluorescence in merged confocal slices of AJ region in wild type (B) and Rho172F MARCM clones coexpressing Nuf (B′). Confocal immunofluorescence localization of Nuf (red) in GFP-labeled Rho172F MARCM clones (C, C′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Representative heat maps of Nuf immunofluorescence in merged confocal slices through the AJ region in wild type (D) and Rho172F MARCM clones (D′). Confocal immunofluorescence localization of Rab11 (green; E, E′, F, F′) and Nuf (red; E, E′′, F, F′′) in AJ region of wild-type PECs (GMR-Gal4; E, E′′) and PECs ubiquitously depleted of Rho1 (GMR-Rho1 RNAi; F, F′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region. White scale bars (lower right corner), 10 μm. (G) Quantitation of percentage vesicles that localize Nuf alone (pink) or those that colocalize Nuf and Rab11 (yellow). (H) Quantitation of percentage vesicles that localize Rab11 alone (green) or those that colocalize Rab11 and Nuf (yellow). The p values were calculated comparing values in GMR-Rho1 RNAi clones (right) to control GMR-Gal4 (left) using an unpaired, two-sided Student's t test; n.s., p > 0.05.
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Figure 4: Rho1 function precedes Rab11 recruitment to recycling endosomes by Nuf. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′) and Rab11 (A′′) in the AJ region of GFP-labeled MARCM Rho172F (Rho1 LoF) PEC clones coexpressing Nuf. Arrowheads indicate rescued AJs. White asterisk denotes wild-type PEC, and yellow asterisk denotes mutant PEC. Representative heat maps of Rab11 immunofluorescence in merged confocal slices of AJ region in wild type (B) and Rho172F MARCM clones coexpressing Nuf (B′). Confocal immunofluorescence localization of Nuf (red) in GFP-labeled Rho172F MARCM clones (C, C′). Images were compiled as a sum of multiple confocal slices within the region where AJs were present. White arrowheads denote wild-type PECs, and yellow arrowheads denote mutant PECs. Representative heat maps of Nuf immunofluorescence in merged confocal slices through the AJ region in wild type (D) and Rho172F MARCM clones (D′). Confocal immunofluorescence localization of Rab11 (green; E, E′, F, F′) and Nuf (red; E, E′′, F, F′′) in AJ region of wild-type PECs (GMR-Gal4; E, E′′) and PECs ubiquitously depleted of Rho1 (GMR-Rho1 RNAi; F, F′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region. White scale bars (lower right corner), 10 μm. (G) Quantitation of percentage vesicles that localize Nuf alone (pink) or those that colocalize Nuf and Rab11 (yellow). (H) Quantitation of percentage vesicles that localize Rab11 alone (green) or those that colocalize Rab11 and Nuf (yellow). The p values were calculated comparing values in GMR-Rho1 RNAi clones (right) to control GMR-Gal4 (left) using an unpaired, two-sided Student's t test; n.s., p > 0.05.
Mentions: Rab11 recruitment to REs is regulated by a family of Rab11-interacting proteins (Rab11-FIPs). In mammals there are two classes of Rab11-FIPs with redundant functions, whereas in Drosophila only two genes have been identified: dRip11, a class I Rab11-FIP, and Nuclear Fallout (Nuf), a class II Rab11-FIP. DRip11 and Nuf are sufficient to increase Rab11 localization and promote RE formation (Shaye et al., 2008; Gault et al., 2012). Therefore we asked whether Rho1 affected the ability of either Drosophila FIP to promote recruitment of Rab11 to vesicles and thereby restore Rab11-positive RE distribution in Rho1-deficient clones. Overexpression of dRip11 restored Rab11 staining pattern in PEC clones devoid of Rho1 (Supplemental Figure S5A′′; quantified in Supplemental Figure S5, B, B′, and C) but was not sufficient to restore AJ organization (Supplemental Figure S5, A and A′; quantified in Supplemental Table S3). On the other hand, Nuf overexpression (Supplemental Figure S5, D and D′) restored both Rab11 staining (Figure 4A′′; quantified in Figure 4, B and B′, and Supplemental Figure S5C) and partially rescued AJs between cells lacking Rho1 (Figure 4, A and A′; quantified in Supplemental Table S3).

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