Limits...
Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension.

Simões Sde M, Mainieri A, Zallen JA - J. Cell Biol. (2014)

Bottom Line: Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling.In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension.These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute and 2 Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065.

ABSTRACT
Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.

Show MeSH

Related in: MedlinePlus

Shroom is required for Rho-kinase localization and force generation during convergent extension. (A–C) Localization of Rho-kinaseK116A and Baz/Par-3 in wild-type (WT; A), ShrmΔ13.6 mutant (B), and ShrmAB (C) RNAi embryos at stage 8. Anterior is left, and ventral is down. (D) Junctional enrichment is the ratio of the mean pixel intensity at adherens junctions divided by the mean pixel intensity in the medial–apical cortex and cytoplasm (pixels ≥1 µm from the cell boundary). Planar polarity is the mean intensity of AP edges (75–90° relative to the AP axis) divided by the mean intensity of DV edges (0–15°). (E) Rho-kinase was less junctionally enriched in Shroom mutants in stage 8 (P < 0.0001). (F) Rho-kinase was less planar polarized in Shroom mutants in stage 8 (P = 0.05). Wild-type Rho-kinase planar polarity was slightly higher than in Fig. 1 C because different Rho-kinaseK116A transgenes and fixation methods were used (Materials and methods). (G and H) Z stack (G) and cross section (H) of Rho-kinaseK116A in a stage 8 wild-type embryo (0 µm is at the level of adherens junctions, and −2 to 8–µm images are at the indicated distance basal to the junctions). (I and J) Z stack (I) and cross section (J) of Rho-kinaseK116A in a stage 8 ShrmΔ11 mutant. (K) Peak retraction velocities after laser ablation of control (flp dsRNA injected) and ShrmAB RNAi embryos in early stage 8. ShrmAB RNAi embryos had slower retraction velocities at AP edges (P = 0.003) and no change at DV edges (P = 0.36) compared with controls (19 AP and 8 DV ablations in flp RNAi; 16 AP and 6 DV ablations in ShrmAB RNAi). ShrmΔ11 and ShrmΔ13.6 were combined for the analysis in E and F. A single value was obtained for each image by analyzing 100–200 edges/image, and 8–11 images in 5–6 embryos were analyzed/genotype. *, P ≤ 0.05. Means ± SEM between images are shown. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3926966&req=5

fig4: Shroom is required for Rho-kinase localization and force generation during convergent extension. (A–C) Localization of Rho-kinaseK116A and Baz/Par-3 in wild-type (WT; A), ShrmΔ13.6 mutant (B), and ShrmAB (C) RNAi embryos at stage 8. Anterior is left, and ventral is down. (D) Junctional enrichment is the ratio of the mean pixel intensity at adherens junctions divided by the mean pixel intensity in the medial–apical cortex and cytoplasm (pixels ≥1 µm from the cell boundary). Planar polarity is the mean intensity of AP edges (75–90° relative to the AP axis) divided by the mean intensity of DV edges (0–15°). (E) Rho-kinase was less junctionally enriched in Shroom mutants in stage 8 (P < 0.0001). (F) Rho-kinase was less planar polarized in Shroom mutants in stage 8 (P = 0.05). Wild-type Rho-kinase planar polarity was slightly higher than in Fig. 1 C because different Rho-kinaseK116A transgenes and fixation methods were used (Materials and methods). (G and H) Z stack (G) and cross section (H) of Rho-kinaseK116A in a stage 8 wild-type embryo (0 µm is at the level of adherens junctions, and −2 to 8–µm images are at the indicated distance basal to the junctions). (I and J) Z stack (I) and cross section (J) of Rho-kinaseK116A in a stage 8 ShrmΔ11 mutant. (K) Peak retraction velocities after laser ablation of control (flp dsRNA injected) and ShrmAB RNAi embryos in early stage 8. ShrmAB RNAi embryos had slower retraction velocities at AP edges (P = 0.003) and no change at DV edges (P = 0.36) compared with controls (19 AP and 8 DV ablations in flp RNAi; 16 AP and 6 DV ablations in ShrmAB RNAi). ShrmΔ11 and ShrmΔ13.6 were combined for the analysis in E and F. A single value was obtained for each image by analyzing 100–200 edges/image, and 8–11 images in 5–6 embryos were analyzed/genotype. *, P ≤ 0.05. Means ± SEM between images are shown. Bars, 10 µm.

Mentions: In wild-type embryos, Rho-kinase is enriched at adherens junctions and is present at lower levels on the lateral membrane (Fig. 4, A and G). In contrast, in Shroom mutants, Rho-kinase accumulated less strongly at adherens junctions and was present in ectopic aggregates in the apical–medial cortex and cytoplasm (Fig. 4, B, E, and I). In addition, Rho-kinase had significantly reduced planar polarity in Shroom mutants compared with wild type (Fig. 4 F). Consistent with the decreased Rho-kinase localization at adherens junctions, the apical enrichment of Rho-kinase was slightly reduced in Shroom mutants (Fig. 4, G–J). Similar defects were observed in ShrmAB RNAi embryos (Fig. 4 C). These results demonstrate that Shroom is required to promote Rho-kinase junctional localization and planar polarity during axis elongation.


Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension.

Simões Sde M, Mainieri A, Zallen JA - J. Cell Biol. (2014)

Shroom is required for Rho-kinase localization and force generation during convergent extension. (A–C) Localization of Rho-kinaseK116A and Baz/Par-3 in wild-type (WT; A), ShrmΔ13.6 mutant (B), and ShrmAB (C) RNAi embryos at stage 8. Anterior is left, and ventral is down. (D) Junctional enrichment is the ratio of the mean pixel intensity at adherens junctions divided by the mean pixel intensity in the medial–apical cortex and cytoplasm (pixels ≥1 µm from the cell boundary). Planar polarity is the mean intensity of AP edges (75–90° relative to the AP axis) divided by the mean intensity of DV edges (0–15°). (E) Rho-kinase was less junctionally enriched in Shroom mutants in stage 8 (P < 0.0001). (F) Rho-kinase was less planar polarized in Shroom mutants in stage 8 (P = 0.05). Wild-type Rho-kinase planar polarity was slightly higher than in Fig. 1 C because different Rho-kinaseK116A transgenes and fixation methods were used (Materials and methods). (G and H) Z stack (G) and cross section (H) of Rho-kinaseK116A in a stage 8 wild-type embryo (0 µm is at the level of adherens junctions, and −2 to 8–µm images are at the indicated distance basal to the junctions). (I and J) Z stack (I) and cross section (J) of Rho-kinaseK116A in a stage 8 ShrmΔ11 mutant. (K) Peak retraction velocities after laser ablation of control (flp dsRNA injected) and ShrmAB RNAi embryos in early stage 8. ShrmAB RNAi embryos had slower retraction velocities at AP edges (P = 0.003) and no change at DV edges (P = 0.36) compared with controls (19 AP and 8 DV ablations in flp RNAi; 16 AP and 6 DV ablations in ShrmAB RNAi). ShrmΔ11 and ShrmΔ13.6 were combined for the analysis in E and F. A single value was obtained for each image by analyzing 100–200 edges/image, and 8–11 images in 5–6 embryos were analyzed/genotype. *, P ≤ 0.05. Means ± SEM between images are shown. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926966&req=5

fig4: Shroom is required for Rho-kinase localization and force generation during convergent extension. (A–C) Localization of Rho-kinaseK116A and Baz/Par-3 in wild-type (WT; A), ShrmΔ13.6 mutant (B), and ShrmAB (C) RNAi embryos at stage 8. Anterior is left, and ventral is down. (D) Junctional enrichment is the ratio of the mean pixel intensity at adherens junctions divided by the mean pixel intensity in the medial–apical cortex and cytoplasm (pixels ≥1 µm from the cell boundary). Planar polarity is the mean intensity of AP edges (75–90° relative to the AP axis) divided by the mean intensity of DV edges (0–15°). (E) Rho-kinase was less junctionally enriched in Shroom mutants in stage 8 (P < 0.0001). (F) Rho-kinase was less planar polarized in Shroom mutants in stage 8 (P = 0.05). Wild-type Rho-kinase planar polarity was slightly higher than in Fig. 1 C because different Rho-kinaseK116A transgenes and fixation methods were used (Materials and methods). (G and H) Z stack (G) and cross section (H) of Rho-kinaseK116A in a stage 8 wild-type embryo (0 µm is at the level of adherens junctions, and −2 to 8–µm images are at the indicated distance basal to the junctions). (I and J) Z stack (I) and cross section (J) of Rho-kinaseK116A in a stage 8 ShrmΔ11 mutant. (K) Peak retraction velocities after laser ablation of control (flp dsRNA injected) and ShrmAB RNAi embryos in early stage 8. ShrmAB RNAi embryos had slower retraction velocities at AP edges (P = 0.003) and no change at DV edges (P = 0.36) compared with controls (19 AP and 8 DV ablations in flp RNAi; 16 AP and 6 DV ablations in ShrmAB RNAi). ShrmΔ11 and ShrmΔ13.6 were combined for the analysis in E and F. A single value was obtained for each image by analyzing 100–200 edges/image, and 8–11 images in 5–6 embryos were analyzed/genotype. *, P ≤ 0.05. Means ± SEM between images are shown. Bars, 10 µm.
Mentions: In wild-type embryos, Rho-kinase is enriched at adherens junctions and is present at lower levels on the lateral membrane (Fig. 4, A and G). In contrast, in Shroom mutants, Rho-kinase accumulated less strongly at adherens junctions and was present in ectopic aggregates in the apical–medial cortex and cytoplasm (Fig. 4, B, E, and I). In addition, Rho-kinase had significantly reduced planar polarity in Shroom mutants compared with wild type (Fig. 4 F). Consistent with the decreased Rho-kinase localization at adherens junctions, the apical enrichment of Rho-kinase was slightly reduced in Shroom mutants (Fig. 4, G–J). Similar defects were observed in ShrmAB RNAi embryos (Fig. 4 C). These results demonstrate that Shroom is required to promote Rho-kinase junctional localization and planar polarity during axis elongation.

Bottom Line: Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling.In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension.These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute and 2 Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065.

ABSTRACT
Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.

Show MeSH
Related in: MedlinePlus