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First published online 1 October 2003
doi: 10.1242/dev.00807

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The Caenorhabditis elegans nonmuscle myosin genes nmy-1 and nmy-2 function as redundant components of the let-502/Rho-binding kinase and mel-11/myosin phosphatase pathway during embryonic morphogenesis

Alisa J. Piekny*, Jacque-Lynne F. Johnson, Gwendolyn D. Cham and Paul E. Mains{dagger}

Genes and Development Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Dr NW, Calgary, AB, Canada, T2N 4N1



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  		Fig. 1. Schematic representation of the predicted NMY-1 structure. The N terminus contains an SH3-like domain and a myosin motor domain, which contains the MLC binding sites (one for the regulatory MLC and one for the essential MLC). The C terminus includes a coiled-coil myosin tail domain, probably involved in MHC dimerization. nmy-1 mutations are indicated. The overall amino acid identities between NMY-1 and Drosophila Zipper, human MHCB and C. elegans NMY-2 are shown as well as the identities between the SH3, myosin motor and myosin tail domains.

 


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  		Fig. 2. Nomarski images of mutant L1 larva. (A) Wild type, (B) mlc-4, (C) let-502(ca201), (D) nmy-1(sb115) and (E,F) nmy-1(sb115); nmy-2(RNAi). (G,H) The adult pharynx of wild-type and nmy-1(sb115) hermaphrodite are indicated by brackets. Scale bars: 10 µm.

 


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  		Fig. 3. NMY-1 and NMY-2 expression in nmy-1(sb115) mutant embryos. nmy-1(sb115) embryos were stained for DAPI (left column), NMY-2 (middle column) and NMY-1 (right column). NMY-2 was expressed prior (B), during (E) and after (H) elongation, although levels decreased. Anti-NMY-1 antisera failed to stain these embryos (C,F,I), with the exception of a low level of crossreactivity in muscle (I). Scale bar: 10 µm.

 


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  		Fig. 4. NMY-1 and actin localization during elongation. Actin (A,C,E) and NMY-1 (B,D,F) localization are shown in deconvolved images of superficial sections of comparably-staged 1.5 to 3.0-fold wild-type embryos using immunofluorescence. Owing to incompatibilities with fixation methods for actin and NMY-1, we could not co-stain embryos. Scale bar: 10 µm.

 


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  		Fig. 5. LET-502 and MEL-11 immunolocalization in wild-type embryos. Embryos in the first column are stained with anti-LET-502, middle column with anti-MEL-11 and merged images are presented in the right column (LET-502 red, MEL-11 green). (A-C) 1.0- to 1.5-fold embryo showing extensive colocalization of LET-502 and MEL-11 in all epidermal cells. (D-F) Twofold embryo is shown at a plane corresponding to the epidermal cells where MEL-11 is restricted to epidermal cell boundaries while LET-502 remains cytoplasmic. (G-I) Twofold embryo at a plane corresponding to pharynx and intestine, where LET-502 and MEL-11 colocalized at adherens junctions. (J-L) Threefold embryo showed cytoplasmic LET-502, while MEL-11 showed punctate staining at the cell boundaries, with a limited amount reappearing in the cytoplasm (compare E and K). All images were deconvolved. Scale bars: 10 µm.

 


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  		Fig. 6. LET-502, but not MEL-11, localizes near NMY-1. In elongating embryos, LET-502 (A) was interspersed with filamentous NMY-1 (B). Merged image is shown in C (LET-502 red, NMY-1 green). A magnified view of C is shown in D. MEL-11 was restricted to epidermal cell boundaries (F) away from NMY-1 (E), with a merged image shown in G (NMY-1 red, MEL-11 green). A magnified view is included in H. All images were deconvolved. Scale bars: 10 µm.

 


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  		Fig. 7. MEL-11 is dependent on LET-502 for its localization. Although only low levels of cytoplasmic anti-MEL-11 staining was present in the lateral epidermal cells of wild type (A), higher cytoplasmic levels were present in let-502(ca201) (B), which arrests at the onset of elongation. let-502(sb106) embryos undergo elongation, albeit abnormally, and also show higher levels of cytoplasmic MEL-11 (C). The let-502(sb106) embryos in D is at a later stage than that in C, and again showed higher levels of cytoplasmic MEL-11 compared with wild type (A). The embryo in D presents a more ventral aspect; the posterior lateral epidermal cells indicated by the bracket were in the focal plane and show increased cytoplasmic MEL-11. All images were deconvolved. Scale bar: 10 µm.

 


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  		Fig. 8. NMY-1 and MEL-11 localization in myosin-depleted embryos. In mlc-4 embryos, NMY-1 collapsed into large foci (A), but MEL-11 was unaffected (B). A merged image is shown in (C, NMY-1 red, MEL-11 green). However, although LET-502 appeared normal (D), MEL-11 was clearly disrupted in nmy-1(sb113); nmy-1(RNAi) embryos (E, merged image shown in F with LET-502 red, MEL-11 green) where it remained cytoplasmic in comparison to its membrane location in wild-type embryos of similar stages (e.g. Fig. 5E). MEL-11 membrane localization was decreased compared with the normal pattern of LET-502 (G), or as shown in this embryo (H), occasionally absent when total NMY was only partially depleted in nmy-1(sb113) (I, merged image, LET-502 red, MEL-11 green). Scale bar: 10 µm.

 


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  		Fig. 9. A cartoon model demonstrating how actin, NMY-1, LET-502 and MEL-11 regulate elongation of lateral epidermal cells. Prior to elongation, actin (red) is disorganized and NMY-1 (black), LET-502 (blue) and MEL-11 (green) are present throughout the cell. In the early stages of elongation, actin filaments organize perpendicular to the direction of the future cell shape changes. NMY-1 begins to organize into filaments, where it could function as a motor to shorten actin filaments. LET-502 remains at high levels within the cell where it likely phosphorylates and thus sequesters MEL-11 to the membrane. By the end of elongation, NMY-1 forms perpendicular lines similar to actin. LET-502 remains associated with NMY-1 and MEL-11 loses its strong membrane localization and partially returns to the cytoplasm, where it could downregulate myosin activity.

 

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© The Company of Biologists Ltd 2003