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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 affects staining of Rab11 recycling endosomes in the AJ region. Schematic representation of apical (left) and lateral (right) views of a pupal eye ommatidium at 41-h APF, identifying the AJ and basal regions used for analyses. Red lines represent AJ-bound DE-cadherin localization, and blue regions correspond to PECs (A). Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; B, B′) and Rab11 (B′′) in AJ region of wild-type PEC clones (white arrowhead) and GFP-labeled Rho1 RNAi PEC clones (yellow arrowhead). Heat maps of sum of multiple confocal slices through the AJ region of representative PECs depicting Rab11 immunofluorescence in representative wild-type (C) and Rho1 RNAi PEC clones (C′). Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of PECs of wild-type PEC (white arrowhead) and PEC clones overexpressing Rho1 (Rho1wt) (yellow arrowhead). Quantitation of the relative Rab11 fluorescence intensity per volume measurement in wild type, Rho172F MARCM PEC clones, and PEC clones depleted of Rho1 (Rho1 RNAi) and overexpressing Rho1 (Rho1wt) (E). The p values were calculated using an unpaired, two-sided Student's t test against values for wild-type PECs. *p < 0.05, **p < 0.01. Confocal immunofluorescence localization of DE-cad (red; F and F′) and Rab5 (endocytic vesicle marker; F′′) in wild-type PEC (white arrowhead) and GFP-labeled Rho172F MARCM clones (yellow arrowhead). Images were compiled as a sum of multiple confocal slices within the region where AJs were present, unless otherwise specified. White scale bars (lower right corner), 10 μm. Western blot analysis for Rab11 and Rho1 expression in w1118 (wild type) and Rho172F heterozygote (Rho172F/+) embryonic lysates. α-Tubulin was used as a loading control (G).
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Figure 2: Rho1 affects staining of Rab11 recycling endosomes in the AJ region. Schematic representation of apical (left) and lateral (right) views of a pupal eye ommatidium at 41-h APF, identifying the AJ and basal regions used for analyses. Red lines represent AJ-bound DE-cadherin localization, and blue regions correspond to PECs (A). Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; B, B′) and Rab11 (B′′) in AJ region of wild-type PEC clones (white arrowhead) and GFP-labeled Rho1 RNAi PEC clones (yellow arrowhead). Heat maps of sum of multiple confocal slices through the AJ region of representative PECs depicting Rab11 immunofluorescence in representative wild-type (C) and Rho1 RNAi PEC clones (C′). Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of PECs of wild-type PEC (white arrowhead) and PEC clones overexpressing Rho1 (Rho1wt) (yellow arrowhead). Quantitation of the relative Rab11 fluorescence intensity per volume measurement in wild type, Rho172F MARCM PEC clones, and PEC clones depleted of Rho1 (Rho1 RNAi) and overexpressing Rho1 (Rho1wt) (E). The p values were calculated using an unpaired, two-sided Student's t test against values for wild-type PECs. *p < 0.05, **p < 0.01. Confocal immunofluorescence localization of DE-cad (red; F and F′) and Rab5 (endocytic vesicle marker; F′′) in wild-type PEC (white arrowhead) and GFP-labeled Rho172F MARCM clones (yellow arrowhead). Images were compiled as a sum of multiple confocal slices within the region where AJs were present, unless otherwise specified. White scale bars (lower right corner), 10 μm. Western blot analysis for Rab11 and Rho1 expression in w1118 (wild type) and Rho172F heterozygote (Rho172F/+) embryonic lysates. α-Tubulin was used as a loading control (G).

Mentions: Endocytosis-recycling assays of DE-cadherin–containing vesicles in live pupal eyes. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′, B, B′), YFP-Rab5 (green; A, A′′, B, B′′), and Rab11 (blue; A, A′′′, B, B′′′) in AJ region of wild-type pupal eyes ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5; A–A′′′) or pupal eyes ubiquitously expressing Rho1 RNAi and YFP-Rab5 (GMR-YFP-Rab5 + Rho1 RNAi; B–B′′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region (Figure 2A and Supplemental Figure S2A). All PECs in this figure and henceforth are 41-h APF. White scale bars (lower right corner), 10 μm. Quantitation of the mean percentage of DE-cad–containing vesicles in the AJ region of wild-type PEC (left) or Rho1 RNAi PEC (right) for YFP-Rab5–positive endocytic vesicles (yellow), YFP-Rab5- and Rab11-positive common recycling endosomes (CRE, blue), Rab11-positive recycling endosomes (pink), or YFP-Rab5- and Rab11-negative vesicles (red) (C). The p values were calculated using an unpaired, two-sided Student's t test against values for GMR-YFP-Rab5 (i.e., wild type) PECs. *p < 0.05, **p < 0.01.


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 affects staining of Rab11 recycling endosomes in the AJ region. Schematic representation of apical (left) and lateral (right) views of a pupal eye ommatidium at 41-h APF, identifying the AJ and basal regions used for analyses. Red lines represent AJ-bound DE-cadherin localization, and blue regions correspond to PECs (A). Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; B, B′) and Rab11 (B′′) in AJ region of wild-type PEC clones (white arrowhead) and GFP-labeled Rho1 RNAi PEC clones (yellow arrowhead). Heat maps of sum of multiple confocal slices through the AJ region of representative PECs depicting Rab11 immunofluorescence in representative wild-type (C) and Rho1 RNAi PEC clones (C′). Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of PECs of wild-type PEC (white arrowhead) and PEC clones overexpressing Rho1 (Rho1wt) (yellow arrowhead). Quantitation of the relative Rab11 fluorescence intensity per volume measurement in wild type, Rho172F MARCM PEC clones, and PEC clones depleted of Rho1 (Rho1 RNAi) and overexpressing Rho1 (Rho1wt) (E). The p values were calculated using an unpaired, two-sided Student's t test against values for wild-type PECs. *p < 0.05, **p < 0.01. Confocal immunofluorescence localization of DE-cad (red; F and F′) and Rab5 (endocytic vesicle marker; F′′) in wild-type PEC (white arrowhead) and GFP-labeled Rho172F MARCM clones (yellow arrowhead). Images were compiled as a sum of multiple confocal slices within the region where AJs were present, unless otherwise specified. White scale bars (lower right corner), 10 μm. Western blot analysis for Rab11 and Rho1 expression in w1118 (wild type) and Rho172F heterozygote (Rho172F/+) embryonic lysates. α-Tubulin was used as a loading control (G).
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Figure 2: Rho1 affects staining of Rab11 recycling endosomes in the AJ region. Schematic representation of apical (left) and lateral (right) views of a pupal eye ommatidium at 41-h APF, identifying the AJ and basal regions used for analyses. Red lines represent AJ-bound DE-cadherin localization, and blue regions correspond to PECs (A). Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; B, B′) and Rab11 (B′′) in AJ region of wild-type PEC clones (white arrowhead) and GFP-labeled Rho1 RNAi PEC clones (yellow arrowhead). Heat maps of sum of multiple confocal slices through the AJ region of representative PECs depicting Rab11 immunofluorescence in representative wild-type (C) and Rho1 RNAi PEC clones (C′). Confocal immunofluorescence localization of DE-cad (red; D, D′) and Rab11 (D′′) in AJ region of PECs of wild-type PEC (white arrowhead) and PEC clones overexpressing Rho1 (Rho1wt) (yellow arrowhead). Quantitation of the relative Rab11 fluorescence intensity per volume measurement in wild type, Rho172F MARCM PEC clones, and PEC clones depleted of Rho1 (Rho1 RNAi) and overexpressing Rho1 (Rho1wt) (E). The p values were calculated using an unpaired, two-sided Student's t test against values for wild-type PECs. *p < 0.05, **p < 0.01. Confocal immunofluorescence localization of DE-cad (red; F and F′) and Rab5 (endocytic vesicle marker; F′′) in wild-type PEC (white arrowhead) and GFP-labeled Rho172F MARCM clones (yellow arrowhead). Images were compiled as a sum of multiple confocal slices within the region where AJs were present, unless otherwise specified. White scale bars (lower right corner), 10 μm. Western blot analysis for Rab11 and Rho1 expression in w1118 (wild type) and Rho172F heterozygote (Rho172F/+) embryonic lysates. α-Tubulin was used as a loading control (G).
Mentions: Endocytosis-recycling assays of DE-cadherin–containing vesicles in live pupal eyes. Confocal immunofluorescence localization of DE-cadherin (DE-cad, red; A, A′, B, B′), YFP-Rab5 (green; A, A′′, B, B′′), and Rab11 (blue; A, A′′′, B, B′′′) in AJ region of wild-type pupal eyes ubiquitously expressing YFP-Rab5 (GMR-YFP-Rab5; A–A′′′) or pupal eyes ubiquitously expressing Rho1 RNAi and YFP-Rab5 (GMR-YFP-Rab5 + Rho1 RNAi; B–B′′′). Images are representative single confocal slices taken in each genotype from a set of slices taken in the AJ region (Figure 2A and Supplemental Figure S2A). All PECs in this figure and henceforth are 41-h APF. White scale bars (lower right corner), 10 μm. Quantitation of the mean percentage of DE-cad–containing vesicles in the AJ region of wild-type PEC (left) or Rho1 RNAi PEC (right) for YFP-Rab5–positive endocytic vesicles (yellow), YFP-Rab5- and Rab11-positive common recycling endosomes (CRE, blue), Rab11-positive recycling endosomes (pink), or YFP-Rab5- and Rab11-negative vesicles (red) (C). The p values were calculated using an unpaired, two-sided Student's t test against values for GMR-YFP-Rab5 (i.e., wild type) PECs. *p < 0.05, **p < 0.01.

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