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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.

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Shroom is required for convergent extension and multicellular rosette formation. (A and B) Stills of time-lapse videos of wild-type (WT; A) and ShrmΔ11 mutant (B) embryos expressing Spider:GFP (t = 0 is the onset of elongation in early stage 7). Stage 7 (0–10 min); stage 8 (10–30 min). Anterior is left, and ventral is down. Rosettes (groups of five or more cells that meet at a vertex) are highlighted. Bar, 10 µm. (C–F) Cell behavior in wild-type (blue) and ShrmΔ11 mutant (red) embryos. (C) Tissue aspect ratio (tissue length along the AP axis relative to its width along the DV axis) normalized to the value at t = 0. Shroom mutants have a reduced tissue aspect ratio in stage 8 (P = 0.02 at 30 min). (D and E) Neighbors lost per cell through neighbor exchange, also known as a T1 process (resulting from single edge contraction events; D) and rosette formation (resulting from the contraction of multiple, consecutive edges; E). Rosette formation was significantly reduced in Shroom mutants (P < 0.001 at 30 min). Local neighbor exchange was not significantly affected (P = 0.63 at 30 min). (F) Mean number of neighbors per cell. Wild-type cells have progressively fewer neighbors midway through elongation as a result of cell rearrangement. Cells in Shroom mutants have more neighbors on average midway through elongation (P < 0.001 at 20 min), consistent with reduced cell rearrangement. Videos of four wild-type Spider:GFP and eight Shroom mutants were analyzed at 15-s intervals (185–276 cells tracked/embryo). Means ± SEM between embryos are shown.
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fig6: Shroom is required for convergent extension and multicellular rosette formation. (A and B) Stills of time-lapse videos of wild-type (WT; A) and ShrmΔ11 mutant (B) embryos expressing Spider:GFP (t = 0 is the onset of elongation in early stage 7). Stage 7 (0–10 min); stage 8 (10–30 min). Anterior is left, and ventral is down. Rosettes (groups of five or more cells that meet at a vertex) are highlighted. Bar, 10 µm. (C–F) Cell behavior in wild-type (blue) and ShrmΔ11 mutant (red) embryos. (C) Tissue aspect ratio (tissue length along the AP axis relative to its width along the DV axis) normalized to the value at t = 0. Shroom mutants have a reduced tissue aspect ratio in stage 8 (P = 0.02 at 30 min). (D and E) Neighbors lost per cell through neighbor exchange, also known as a T1 process (resulting from single edge contraction events; D) and rosette formation (resulting from the contraction of multiple, consecutive edges; E). Rosette formation was significantly reduced in Shroom mutants (P < 0.001 at 30 min). Local neighbor exchange was not significantly affected (P = 0.63 at 30 min). (F) Mean number of neighbors per cell. Wild-type cells have progressively fewer neighbors midway through elongation as a result of cell rearrangement. Cells in Shroom mutants have more neighbors on average midway through elongation (P < 0.001 at 20 min), consistent with reduced cell rearrangement. Videos of four wild-type Spider:GFP and eight Shroom mutants were analyzed at 15-s intervals (185–276 cells tracked/embryo). Means ± SEM between embryos are shown.

Mentions: The failure to maintain Rho-kinase and myosin II localization at specific planar junctions is predicted to impair the generation of sustained actomyosin contractility during cell rearrangement. To test whether Shroom is required for cell behavior, we performed time-lapse imaging of wild-type and Shroom mutant embryos expressing Spider:GFP (Videos 7 and 8). During axis elongation, the Drosophila germband elongates along the AP axis and simultaneously narrows along the DV axis, producing a 4.00 ± 0.32–fold increase in the tissue aspect ratio (length to width ratio), a process known as convergent extension. Shroom mutants had reduced convergent extension, resulting in a smaller increase in the tissue length to width ratio compared with wild type (3.16 ± 0.15–fold, P = 0.02; Fig. 6 C). In contrast, tissue length along the AP axis was only slightly reduced in Shroom mutants (1.99 ± 0.04–fold) compared with wild type (2.13 ± 0.05–fold, P = 0.074). These results show that Shroom is required for convergent extension but does not strongly affect the final length of the tissue.


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 convergent extension and multicellular rosette formation. (A and B) Stills of time-lapse videos of wild-type (WT; A) and ShrmΔ11 mutant (B) embryos expressing Spider:GFP (t = 0 is the onset of elongation in early stage 7). Stage 7 (0–10 min); stage 8 (10–30 min). Anterior is left, and ventral is down. Rosettes (groups of five or more cells that meet at a vertex) are highlighted. Bar, 10 µm. (C–F) Cell behavior in wild-type (blue) and ShrmΔ11 mutant (red) embryos. (C) Tissue aspect ratio (tissue length along the AP axis relative to its width along the DV axis) normalized to the value at t = 0. Shroom mutants have a reduced tissue aspect ratio in stage 8 (P = 0.02 at 30 min). (D and E) Neighbors lost per cell through neighbor exchange, also known as a T1 process (resulting from single edge contraction events; D) and rosette formation (resulting from the contraction of multiple, consecutive edges; E). Rosette formation was significantly reduced in Shroom mutants (P < 0.001 at 30 min). Local neighbor exchange was not significantly affected (P = 0.63 at 30 min). (F) Mean number of neighbors per cell. Wild-type cells have progressively fewer neighbors midway through elongation as a result of cell rearrangement. Cells in Shroom mutants have more neighbors on average midway through elongation (P < 0.001 at 20 min), consistent with reduced cell rearrangement. Videos of four wild-type Spider:GFP and eight Shroom mutants were analyzed at 15-s intervals (185–276 cells tracked/embryo). Means ± SEM between embryos are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Shroom is required for convergent extension and multicellular rosette formation. (A and B) Stills of time-lapse videos of wild-type (WT; A) and ShrmΔ11 mutant (B) embryos expressing Spider:GFP (t = 0 is the onset of elongation in early stage 7). Stage 7 (0–10 min); stage 8 (10–30 min). Anterior is left, and ventral is down. Rosettes (groups of five or more cells that meet at a vertex) are highlighted. Bar, 10 µm. (C–F) Cell behavior in wild-type (blue) and ShrmΔ11 mutant (red) embryos. (C) Tissue aspect ratio (tissue length along the AP axis relative to its width along the DV axis) normalized to the value at t = 0. Shroom mutants have a reduced tissue aspect ratio in stage 8 (P = 0.02 at 30 min). (D and E) Neighbors lost per cell through neighbor exchange, also known as a T1 process (resulting from single edge contraction events; D) and rosette formation (resulting from the contraction of multiple, consecutive edges; E). Rosette formation was significantly reduced in Shroom mutants (P < 0.001 at 30 min). Local neighbor exchange was not significantly affected (P = 0.63 at 30 min). (F) Mean number of neighbors per cell. Wild-type cells have progressively fewer neighbors midway through elongation as a result of cell rearrangement. Cells in Shroom mutants have more neighbors on average midway through elongation (P < 0.001 at 20 min), consistent with reduced cell rearrangement. Videos of four wild-type Spider:GFP and eight Shroom mutants were analyzed at 15-s intervals (185–276 cells tracked/embryo). Means ± SEM between embryos are shown.
Mentions: The failure to maintain Rho-kinase and myosin II localization at specific planar junctions is predicted to impair the generation of sustained actomyosin contractility during cell rearrangement. To test whether Shroom is required for cell behavior, we performed time-lapse imaging of wild-type and Shroom mutant embryos expressing Spider:GFP (Videos 7 and 8). During axis elongation, the Drosophila germband elongates along the AP axis and simultaneously narrows along the DV axis, producing a 4.00 ± 0.32–fold increase in the tissue aspect ratio (length to width ratio), a process known as convergent extension. Shroom mutants had reduced convergent extension, resulting in a smaller increase in the tissue length to width ratio compared with wild type (3.16 ± 0.15–fold, P = 0.02; Fig. 6 C). In contrast, tissue length along the AP axis was only slightly reduced in Shroom mutants (1.99 ± 0.04–fold) compared with wild type (2.13 ± 0.05–fold, P = 0.074). These results show that Shroom is required for convergent extension but does not strongly affect the final length of the tissue.

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