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Rho-binding kinase (LET-502) and myosin phosphatase (MEL-11) regulate cytokinesis in the early Caenorhabditis elegans embryo

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

View larger version (95K): [in a new window]   Fig. 1. let-502(sb106) and mel-11(it26) embryos display defects during pseudocleavage and early cleavages. (A-F) A wild-type embryo during pseudocleavage, pronuclear fusion, late first cell division, two-cell stage, late second division and four-cell stage, respectively. (G-L) A mel-11(it26) embryo during similar stages. White arrows indicate ectopic furrows. (M-R) let-502(sb106) embryo at stages similar to the wild-type embryo in (A-F). Pseudocleavage furrows either do not form or are small in comparison with wildtype (compare A with M). Embryos that have unsuccessful cleavages still form short furrows as indicated by the black arrowhead (O), but these regress. This particular embryo underwent successful cleavages during the next round of cell division, forming an abnormal four-cell embryo (R). Wild-type (S), mel-11(it26) (U) and let-502(sb106) (V) embryo dividing from the two- to the four-cell stage. All are at the same cell cycle stage as judged by nuclear and spindle morphology and the time since the previous division. Arrows indicate the AB cell furrow, which completes cleavage early in mel-11(it26) and late in let-502(sb106) relative to wildtype. Bars, 7 µm.

View larger version (26K): [in a new window]   Fig. 2. Furrow ingression times for two- to four-cell stage embryos undergoing AB (left) and P1 (right) cell divisions. AB and P1 cell cleavage furrows that ingress slower in let-502(sb106) were normal in let-502(sb108) but were faster in mel-11(it26) embryos. let-502(sb108 or sb106) and mel-11(it26) suppresses one another's cleavage defects in double mutants, resulting in furrow ingression times closer to wildtype. Error bars show 1 standard deviation. Failed cell divisions are not included in these data. Wildtype: n=7; let-502(sb106): n=9; let-502(sb108): n=9; mel-11(it26): n=9; let-502(sb108) mel-11(it26): n=10; 502(sb106) mel-11(it26): n=6.

View larger version (58K): [in a new window]   Fig. 3. LET-502 and MEL-11 localize at cleavage furrows. (i) IF of LET-502, MEL-11 and DAPI in wild-type, let-502(sb106) or mel-11(it26) embryos. (A-C) Arrows indicate that both LET-502 and MEL-11 are enriched at the furrow during early stages of wild-type cleavage and are found throughout the furrow as it ingresses (D-F). They remain at cell boundaries after cell divisions are completed. (G,I) Cytoplasmic LET-502 is decreased in let-502(sb106) mutant embryos. (H,I) MEL-11 staining is weakened in let-502(sb106) in comparison with wild-type embryos and is almost entirely depleted in mel-11(it26) embryos (K,L); however, LET-502 retained its location (J,L) in mel-11 mutants. Bar, 9 µm. (A-F) n=21, (G,I) n=52, (H,I) n=40, (K,L) n=15, (J,L) n=38. (ii) LET-502 and MEL-11 localization in wild-type and let-502(sb106) embryos using digital deconvolution microscopy. Arrows indicate the ingressing cleavage furrows. (A-C) LET-502 and MEL-11 overlap at the ingressing furrow in wild-type, let-502(sb106) (D-F) and embryos from let-502(ca201)/+ hermaphrodites (G-I). Bar, 6.5 µm. (A-C) n=7, (D-F) n=4 and (G-I) n=4.

View larger version (71K): [in a new window]   Fig. 5. Localization of active rMLC and LET-502 in embryos using digital deconvolution microscopy. All images shown are merged with rMLC posphoserine 19/18 in green, LET-502 in red and DAPI in blue, and arrows point to ingressing cleavage furrows. (A) rMLC phosphoserine 19/18 and LET-502 colocalize in wild-type embryos. (B-C) The intensity of rMLC posphoserine 19/18 decreases in let-502(sb106) embryos and increases in mel-11(it26) embryos in comparison with wild-type embryos. (D,E) rMLC phosphoserine 19/18 is not detectable or is present at low levels in mlc-4(RNAi) embryos. (F) rMLC phosphoserine 19/18 levels are restored in let-502(sb106RNAi); mel-11(it26) embryos. Bar, 6 µm. (A) n=4, (B) n=4, (C) n=2, (D) n=1, (E) n=1 and (F) n=6.

View larger version (52K): [in a new window]   Fig. 7. CYK-1 is dependent on LET-502 but not on MEL-11 for furrow localization. (A-C) CYK-1 colocalized with LET-502 in wild-type embryos, but its localization was partially disrupted in let-502(sb106RNAi) embryos (D-F). (G-I) CYK-1 retained its localization in mel-11(it26) embryos. Bar, 6 µm. (A-C) n=2 by deconvolution and n=4 by IF, (D-F) n=3 by deconvolution and n=2 by IF and (G-I) n=1 by deconvolution.

View larger version (63K): [in a new window]   Fig. 4. Localization of LET-502 with myosin (NMY-2) in wild-type embryos, and localization of NMY-2 and actin in let-502 and mel-11 mutant embryos using digital deconvolution microscopy. Arrows indicate the ingressing or newly completed cleavage furrows. (A-C) LET-502 and NMY-2 colocalize at the ingressing furrow during cleavage. (D-I) NMY-2 and actin still accumulate and colocalize to the putative site of furrow formation in all of the let-502(sb106) and let-502(sb106RNAi) embryos examined. Note that NMY-2 also localizes to the spindle during metaphase (G). NMY-2 and actin (J-L) and LET-502 and NMY-2 (M-O) retain their localization at the furrow in a mel-11(it26) mutant embryo during cleavage. (A-C) Bar, 5 µm. n=3 by deconvolution and n=13 by IF. (D-I) Bar, 7 µm. n=11 let-502(sb106RNAi) stained for both NMY-2 and actin by deconvolution and n=5 let-502(sb106RNAi) embryos stained for NMY-2 only by IF. (J-L) n=4 stained for both NMY-2 and actin by deconvolution, n=23 stained for NMY-2 only by IF and n=2 stained for actin only by IF. (M-O) n=2 by deconvolution and n=23 by IF.

View larger version (71K): [in a new window]   Fig. 6. LET-502 and MEL-11 require MLC-4 and early CYK-1 function for their localization but do not require late CYK-1 function or CYK-4. (A-C) LET-502 and MEL-11 no longer localize to the site of cleavage furrow formation in cyk-1(s2833/t1568) embryos, which show the early cyk-1 phenotype, or in mlc-4(RNAi) embryos (D-F). (G-I) LET-502 and MEL-11 retain their localization in cyk-4(t1689) embryos. (J-L) LET-502 and MEL-11 retain their localization in cyk-1(t1568) embryos, which display the late cyk-1 phenotype. cyk-1(t1611) embryos gave similar results (data not shown). Bar, 6 µm. (A-C) n=5 by IF, (D-F) n=10 by IF, (G-I) n=7 by deconvolution and n=6 by IF and (J-L) n=8 by deconvolution.

View larger version (49K): [in a new window]   Fig. 8. let-502 affects oocyte cellularization, and LET-502 and MEL-11 localize to gonad membranes. (i) A schematic outlines the structure of a gonad arm from an adult hermaphrodite. (ii) Nomarski photograph of two embryos of very different sizes collected from a let-502(sb106) hermaphrodite. Compared with wildtype, which has a very narrow size distribution, let-502 embryos vary substantially above and below the mean (data not shown). Bar, 7.5 µm. (iii) The distal portion of the arm, consisting of a syncitium of nuclei in meiotic prophase, was immunostained for deconvolution. Partitions outline each nucleus (as observed from z sections taken superficially near the surface of the gonad arm in A-C and G-I), but these partitions do not extend into the central core (as observed by more central z sections D-F and J-L) where the nuclei are connected by a common cytoplasm. (A-F) LET-502 and MEL-11 colocalize at the membrane that surrounds the syncitial nuclei in wild-type gonads. (A-C) is a superficial z section and (D-F) is a z section from near the centre of the arm. (G-L) Gonads from let-502(sb106) hermaphrodites are thinner compared with a corresponding region of the wild-type gonad and are sometimes multinucleate, as indicated by the arrow (G-I superficial view versus J-L central view). Bar, 10 µm. (iv) (M-Q) LET-502, NMY-2 and MEL-11 localize to oocyte boundaries after cellularization. Bar, 25 µm. (A-F) n=4, (M-O) n=6 stained for both LET-502 and NMY-2 and n=23 stained for LET-502 only, (P-Q) n=8.

View larger version (26K): [in a new window]   Fig. 9. Pathway showing how let-502 and mel-11 act with respect to other genes to regulate cytokinesis. During anaphase, structural components of the furrow begin to associate into a contractile bundle, including NMY-2, MLC-4 and actin. Cleavage furrow contraction is regulated by MLC-4 phosphorylation. LET-502 and MEL-11 localize to the furrow and together regulate the rate of its contraction. MEL-11 would prevent cleavage furrow contraction from occurring until the proper signal (Rho-GTP) is produced to increase LET-502's activity. Active LET-502 causes the downregulation of MEL-11 and/or the direct activation of MLC-4, allowing the actomyosin ring to contract. After the ring contracts, proteins such as CYK-4 and late CYK-1 regulate furrow termination.

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