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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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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.
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Figure 1: 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.

Mentions: To determine whether Rho1 affected postendocytic routes of DE-cadherin trafficking, we adapted a DE-cadherin endocytosis-recycling assay (Langevin et al., 2005) to live Drosophila pupal eyes. To label the pool of DE-cadherin at AJs but not within cytosolic or biosynthetic pools, we dissected pupal eyes ubiquitously expressing yellow fluorescent protein (YFP)–Rab5 (GMR-YFP-Rab5) or also ubiquitously depleted of Rho1 (GMR-YFP-Rab5 + Rho1 RNA interference [RNAi]) and immediately incubated them at 4°C in medium containing an antibody specific to the extracellular domain of DE-cadherin. Because the apical region of the pupal eye epithelium is unobstructed by cuticles, and septate junctions lie basal to AJs in Drosophila, AJs are accessible to the DE-cadherin antibody. After washing away unbound antibody, we incubated tissues at 25°C for 2 h to allow for endocytosis and recycling of AJ-localized DE-cadherin. Tissues were then fixed, permeabilized, and stained with a Rab11 antibody, and colocalization of internalized DE-cadherin to various endocytic vesicles was measured. To enumerate vesicle staining and control against redundancy in quantification, each captured confocal slice (0.47 μm) through the AJ region of PEC (seven slices: apical to basal) was individually and sequentially analyzed in ImageJ (Supplemental Figure S2A). We scored the percentage of DE-cadherin–positive vesicles that were Rab5 positive (endocytic vesicles), Rab5 and Rab11 positive (common recycling endosomes or CREs), and Rab11 positive (recycling endosomes or REs; Figure 1, A–A′′′and B–B′′′, and Supplemental Figure S2A). In control experiments, expression of YFP-Rab5 did not increase the number of DE-cadherin–positive vesicles detected (Supplemental Figure S2B). Consistent with the effect of Rho1 depletion on increased DE-cadherin endocytosis, the average number of DE-cadherin–positive vesicles that colocalized with Rab5 in endocytic vesicles and in Rab5- and Rab11-positive CREs was significantly increased in Rho1-depleted cells as compared with wild-type cells (Supplemental Figure S2C). In contrast, the average number of DE-cadherin–positive vesicles in Rab11-positive REs was not significantly different between Rho1-depleted cells and wild-type cells. Because the total number of DE-cadherin–positive vesicles was increased in Rho1-depleted PECs compared with wild type, we calculated whether the proportion of DE-cadherin–positive vesicles that resided in each vesicular compartment was altered. In wild-type and Rho1-depleted PECs, there were comparable proportions of DE-cadherin–positive vesicles that did not stain with Rab5 or Rab11, as well as Rab5-positive, DE-cadherin–containing endocytic vesicles (Figure 1C). However, in pupal eyes depleted of Rho1, there was a significant increase in the proportion of DE-cadherin–positive vesicles characteristic of CREs (Rab5 and Rab11 positive) and a decrease in the proportion of DE-cadherin–positive REs (Rab11 positive; Figure 1C). Images shown are representative of individual slices captured within the AJ region of PECs. This analysis in live tissue suggested that Rho1 activity contributed to the formation or stability of DE-cadherin–containing, Rab11-positive vesicles, possibly from the CRE pool.


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)

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.
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Figure 1: 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.
Mentions: To determine whether Rho1 affected postendocytic routes of DE-cadherin trafficking, we adapted a DE-cadherin endocytosis-recycling assay (Langevin et al., 2005) to live Drosophila pupal eyes. To label the pool of DE-cadherin at AJs but not within cytosolic or biosynthetic pools, we dissected pupal eyes ubiquitously expressing yellow fluorescent protein (YFP)–Rab5 (GMR-YFP-Rab5) or also ubiquitously depleted of Rho1 (GMR-YFP-Rab5 + Rho1 RNA interference [RNAi]) and immediately incubated them at 4°C in medium containing an antibody specific to the extracellular domain of DE-cadherin. Because the apical region of the pupal eye epithelium is unobstructed by cuticles, and septate junctions lie basal to AJs in Drosophila, AJs are accessible to the DE-cadherin antibody. After washing away unbound antibody, we incubated tissues at 25°C for 2 h to allow for endocytosis and recycling of AJ-localized DE-cadherin. Tissues were then fixed, permeabilized, and stained with a Rab11 antibody, and colocalization of internalized DE-cadherin to various endocytic vesicles was measured. To enumerate vesicle staining and control against redundancy in quantification, each captured confocal slice (0.47 μm) through the AJ region of PEC (seven slices: apical to basal) was individually and sequentially analyzed in ImageJ (Supplemental Figure S2A). We scored the percentage of DE-cadherin–positive vesicles that were Rab5 positive (endocytic vesicles), Rab5 and Rab11 positive (common recycling endosomes or CREs), and Rab11 positive (recycling endosomes or REs; Figure 1, A–A′′′and B–B′′′, and Supplemental Figure S2A). In control experiments, expression of YFP-Rab5 did not increase the number of DE-cadherin–positive vesicles detected (Supplemental Figure S2B). Consistent with the effect of Rho1 depletion on increased DE-cadherin endocytosis, the average number of DE-cadherin–positive vesicles that colocalized with Rab5 in endocytic vesicles and in Rab5- and Rab11-positive CREs was significantly increased in Rho1-depleted cells as compared with wild-type cells (Supplemental Figure S2C). In contrast, the average number of DE-cadherin–positive vesicles in Rab11-positive REs was not significantly different between Rho1-depleted cells and wild-type cells. Because the total number of DE-cadherin–positive vesicles was increased in Rho1-depleted PECs compared with wild type, we calculated whether the proportion of DE-cadherin–positive vesicles that resided in each vesicular compartment was altered. In wild-type and Rho1-depleted PECs, there were comparable proportions of DE-cadherin–positive vesicles that did not stain with Rab5 or Rab11, as well as Rab5-positive, DE-cadherin–containing endocytic vesicles (Figure 1C). However, in pupal eyes depleted of Rho1, there was a significant increase in the proportion of DE-cadherin–positive vesicles characteristic of CREs (Rab5 and Rab11 positive) and a decrease in the proportion of DE-cadherin–positive REs (Rab11 positive; Figure 1C). Images shown are representative of individual slices captured within the AJ region of PECs. This analysis in live tissue suggested that Rho1 activity contributed to the formation or stability of DE-cadherin–containing, Rab11-positive vesicles, possibly from the CRE pool.

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