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The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Choi W, Jung KC, Nelson KS, Bhat MA, Beitel GJ, Peifer M, Fanning AS - Mol. Biol. Cell (2011)

Bottom Line: Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure.The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together.Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, USA.

ABSTRACT
Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of alleles. We generated alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

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Both dorsal closure and zippering are significantly slowed in pydMZ mutants. (A, B) Stills from a movie of representative wild-type or pydMZ mutant embryos expressing the actin-binding domain of moesin fused to GFP (moe-GFP). Videos are included as Supplemental Figure 5A.mov and Supplemental Figure 5B.mov. Times are indicated, beginning when maximum width of the dorsal opening was 60 μm. Double-headed arrows indicate distance between the two canthi, which is reduced by zippering. Arrows in B indicates examples of wavy margin of the epidermal sheet of cells. (C) Quantitation of time of closure, beginning when maximum width of the dorsal opening was 60 μm. D. Quantitation of the rate of zippering. At t = 0, the position of the anterior canthus was noted, and Δd was calculated as the distance in microns from this position to the position of the anterior canthus at the time indicated. (E, F) Close-ups of leading edge in wild-type (E) and mutant (F) embryos. Arrows indicate wavy leading edge.
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Figure 5: Both dorsal closure and zippering are significantly slowed in pydMZ mutants. (A, B) Stills from a movie of representative wild-type or pydMZ mutant embryos expressing the actin-binding domain of moesin fused to GFP (moe-GFP). Videos are included as Supplemental Figure 5A.mov and Supplemental Figure 5B.mov. Times are indicated, beginning when maximum width of the dorsal opening was 60 μm. Double-headed arrows indicate distance between the two canthi, which is reduced by zippering. Arrows in B indicates examples of wavy margin of the epidermal sheet of cells. (C) Quantitation of time of closure, beginning when maximum width of the dorsal opening was 60 μm. D. Quantitation of the rate of zippering. At t = 0, the position of the anterior canthus was noted, and Δd was calculated as the distance in microns from this position to the position of the anterior canthus at the time indicated. (E, F) Close-ups of leading edge in wild-type (E) and mutant (F) embryos. Arrows indicate wavy leading edge.

Mentions: To further characterize Pyd's role in dorsal closure, we examined the process live in wild-type and pydMZ mutant embryos, using moesin-GFP to visualize F-actin and cell shapes. Dorsal closure in pydMZ mutants was significantly slower than in wild-type embryos (Figure 5, A vs. B; Video 1 vs. Video 2). To quantify this, we assessed the time required to close, beginning when the greatest distance between the two epidermal sheets was 60 μm. Closure was >50% slower in pydMZ mutants (Figure 5C). Many of the morphogenesis defects we observed in fixed embryos were also readily apparent in our movies. The leading edge was very uneven (Figure 5, A and E, vs. B and F, arrows), and the delay in zippering at the canthi, leading to a more elongated dorsal opening (Figure 5, A vs. B, double-headed arrows), was also apparent in the movies. To quantify the delay in zippering, we measured the rate of zippering from a fixed point representing the position of the anterior canthus when the two epidermal sheets were 60 μm apart. This revealed that the zippering rate was significantly slower in pydMZ mutants (Figure 5D), consistent with the defects we saw in fixed images. Thus Pyd is essential for both the normal kinetics and cellular dynamics of dorsal closure, but the process is robust enough that closure is completed in most embryos in its absence.


The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled.

Choi W, Jung KC, Nelson KS, Bhat MA, Beitel GJ, Peifer M, Fanning AS - Mol. Biol. Cell (2011)

Both dorsal closure and zippering are significantly slowed in pydMZ mutants. (A, B) Stills from a movie of representative wild-type or pydMZ mutant embryos expressing the actin-binding domain of moesin fused to GFP (moe-GFP). Videos are included as Supplemental Figure 5A.mov and Supplemental Figure 5B.mov. Times are indicated, beginning when maximum width of the dorsal opening was 60 μm. Double-headed arrows indicate distance between the two canthi, which is reduced by zippering. Arrows in B indicates examples of wavy margin of the epidermal sheet of cells. (C) Quantitation of time of closure, beginning when maximum width of the dorsal opening was 60 μm. D. Quantitation of the rate of zippering. At t = 0, the position of the anterior canthus was noted, and Δd was calculated as the distance in microns from this position to the position of the anterior canthus at the time indicated. (E, F) Close-ups of leading edge in wild-type (E) and mutant (F) embryos. Arrows indicate wavy leading edge.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Both dorsal closure and zippering are significantly slowed in pydMZ mutants. (A, B) Stills from a movie of representative wild-type or pydMZ mutant embryos expressing the actin-binding domain of moesin fused to GFP (moe-GFP). Videos are included as Supplemental Figure 5A.mov and Supplemental Figure 5B.mov. Times are indicated, beginning when maximum width of the dorsal opening was 60 μm. Double-headed arrows indicate distance between the two canthi, which is reduced by zippering. Arrows in B indicates examples of wavy margin of the epidermal sheet of cells. (C) Quantitation of time of closure, beginning when maximum width of the dorsal opening was 60 μm. D. Quantitation of the rate of zippering. At t = 0, the position of the anterior canthus was noted, and Δd was calculated as the distance in microns from this position to the position of the anterior canthus at the time indicated. (E, F) Close-ups of leading edge in wild-type (E) and mutant (F) embryos. Arrows indicate wavy leading edge.
Mentions: To further characterize Pyd's role in dorsal closure, we examined the process live in wild-type and pydMZ mutant embryos, using moesin-GFP to visualize F-actin and cell shapes. Dorsal closure in pydMZ mutants was significantly slower than in wild-type embryos (Figure 5, A vs. B; Video 1 vs. Video 2). To quantify this, we assessed the time required to close, beginning when the greatest distance between the two epidermal sheets was 60 μm. Closure was >50% slower in pydMZ mutants (Figure 5C). Many of the morphogenesis defects we observed in fixed embryos were also readily apparent in our movies. The leading edge was very uneven (Figure 5, A and E, vs. B and F, arrows), and the delay in zippering at the canthi, leading to a more elongated dorsal opening (Figure 5, A vs. B, double-headed arrows), was also apparent in the movies. To quantify the delay in zippering, we measured the rate of zippering from a fixed point representing the position of the anterior canthus when the two epidermal sheets were 60 μm apart. This revealed that the zippering rate was significantly slower in pydMZ mutants (Figure 5D), consistent with the defects we saw in fixed images. Thus Pyd is essential for both the normal kinetics and cellular dynamics of dorsal closure, but the process is robust enough that closure is completed in most embryos in its absence.

Bottom Line: Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure.The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together.Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, USA.

ABSTRACT
Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of alleles. We generated alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

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