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First published online 28 September 2004
doi: 10.1242/jcs.01389

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Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo

Torah Kachur^*, Wanyuan Ao^*,, Jeffrey Berger and Dave Pilgrim^¶

Department of Biological Sciences, CW-405 Biological Sciences Building, University of Alberta, Edmonton, T6G 2E9, Canada

View larger version (53K): [in a new window]   Fig. 1. UNC-45 expression in the nematode Caenorhabditis elegans adult germline and early embryo. (A) A hermaphrodite gonad doubly stained with anti-UNC-45 antibody (green) and DAPI (blue), which stains the cell nuclei. (B) Distal part of hermaphrodite gonad doubly stained with pre-immune serum and DAPI as controls. (C,D) Two-cell (C) and 16-cell (D) embryos stained with anti-UNC-45. Arrows indicate concentration of UNC-45 at cell boundaries. In panels E-H, embryos are doubly stained for UNC-45 (E,G) and monoclonal MH27 (F,H), which recognizes an epitope associated with adherent junctions at the boundaries of hypodermal cells (Francis and Waterston, 1985; Hresko et al., 1994). As the embryo begins to elongate, the UNC-45 staining is progressively restricted to the presumptive muscle cells (G, arrowhead). (I,J) GFP and DIC images of a two-fold embryo transgenic for an UNC-45::GFP fusion protein, showing expression in the body wall muscles (arrowhead). (K-N) UNC-45 staining is detectable throughout embryogenesis whereas MHC-B staining is only detectable in the developing body wall muscles. K and M are stained with anti-UNC-45, L and N with anti-MHC B (5-8). (K,L) The same embryo at about 250 minutes after fertilization. (M,N) The same embryo at the 1.5-fold stage of elongation. The images of L and N were exposed for the same times, serving as controls to show that although MHC B does not show staining in the early embryos, UNC-45 does.

View larger version (128K): [in a new window]   Fig. 2. Cytokinesis defects in C. elegans embryos derived from mothers of the genotype unc-45(st601let) /unc-45(r450ts) carrying an extrachromosomal array with the wild-type unc-45 cDNA fused to GFP, raised at 25°C. (A-E) Time course shows the same embryo photographed at different times. Complete cytokinesis was not observed, although many attempts were made. Times are relative to the beginning of observation: (A) 0 minutes; (B) 15 minutes; (C) 25 minutes; (D) 40 minutes; (E) 70 minutes. (F) Embryo from a different mother of the same genotype. Note the multiple nuclei in one cell (arrowhead). (G-I) A wild-type embryo is normally able to complete at least two cell divisions in a similar time. (G) 0 minutes, pronuclear meeting; (H) 4 minutes, first cleavage; (I) 14 minutes, second cleavage. Embryos were observed under differential interference contrast optics.

View larger version (42K): [in a new window]   Fig. 3. Characterization of isolated clones from a yeast two-hybrid screening of a C. elegans cDNA library with unc-45 cDNA. (A) Two of the clones identified from yeast two-hybrid screens. UNC-45 from a full-length cDNA (pDP#WA036) interacts with the products of fragments of two different myosin cDNAs, NMY-2 and HUM-2. The TPR domain alone was tested and showed no interaction with either NMY-2 or HUM-2. (B) Quantitative ß-gal assay for the yeast two-hybrid clones. (C) Diagram of a conventional myosin II molecule, showing the ATP and actin binding sites in the head domain, IQ motif (light chain and calmodulin binding sequence) in the neck domain and the coiled-coil tail domain. Schematic diagram of MHC B and two myosins identified from the yeast two-hybrid screens that interact with UNC-45. HUM-2 is a type V myosin and the identified cDNA fragment encodes a 140 amino acid segment located in the head domain (striped shading). NMY-2 is a non-muscle type II myosin and a region of 530 amino acids (striped shading) located mostly in the head domain interacts with UNC-45. Note that these two identified regions overlap in the highly conserved head domain as indicated. In UNC-54, the portion of the molecule that is sufficient to localize UNC-45 to the body wall muscle thick filaments is indicated by checked shading. Detailed domain analysis of NMY-2 is not available.

View larger version (111K): [in a new window]   Fig. 4. Immunolocalization of UNC-45 and NMY-2 in wild-type and nmy-2(RNAi) embryos. (A-F) Wild-type embryos of increasing age stained with anti-UNC-45 (green) and anti-NMY-2 (red) to show the concentration of UNC-45 and NMY-2 at the cell boundaries. (A-C) Two-cell embryo with the merged image showing colocalization of UNC-45 and NMY-2. (D-F) 20-cell stage embryo with merged image. Images G and H show embryos treated with RNA-mediated interference (RNAi) against NMY-2, stained with anti-UNC-45 showing that the concentration of UNC-45 at the cell boundaries is disrupted when NMY-2 is knocked down.