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A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the early C. elegans embryo

¹ Research Institute for Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria
² Technical University Braunschweig, D-38106, Braunschweig, Germany
³ Max Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany

View larger version (104K): [in a new window]   Fig. 1. cyk-3 mutant embryos fail to perform cytokinesis. (a) cyk-3 embryos were dissected from homozygous mutant hermaphrodites, and time-lapse microscopy was performed. During pronuclear migration, cyk-3 embryos do not undergo any visible membrane contractions, and they fail to perform pseudocleavage. At the time of anaphase, when wild-type embryos initiate cytokinesis, cyk-3 embryos show no sign of furrowing. At that stage, the mitotic spindle of both wild-type and cyk-3 embryos is displaced towards the posterior hemisphere of the cell. While nuclear division occurs with normal kinetics, cytokinesis does not occur in these embryos. (b) Time-lapse analysis was performed on embryos within the uterus of homozygous mutant hermaphrodites. As dissected embryos, embryos inside the uterus do not show any furrowing at the time of anaphase. Nuclear division, however, occurs normally.

View larger version (58K): [in a new window]   Fig. 2. P-granules are mislocalized in cyk-3 mutant embryos. Embryos were dissected from homozygous mutant or wild-type hermaphrodites, fixed and stained with anti-tubulin (green), anti-PGL-1 (red) and Hoechst 33342 (blue). (a) In wild-type embryos, P-granules are localized to the posterior cortex during anaphase. (b) In cyk-3 mutant embryos, P-granules are not localized to the posterior cortex during anaphase.

View larger version (96K): [in a new window]   Fig. 3. cyk-3 embryos fail to redistrubute f-actin but form an actin ring at the presumptive cleavage site. Embyros from either homozygous mutant or wild-type hermaphrodites were dissected directly into fixative containing rhodamine-labeled Phalloidin. DNA is stained with Hoechst 33342 and shown in the small insets. (a) At pronuclear meeting, the actin cytoskeleton of wild-type embryos is confined to the anterior hemisphere, forming the `anterior cap' (surface view). In cyk-3 mutant embryos of the same stage, the actin is homogeneously distributed (surface view). (b) In wild-type embryos at late telophase, a prominent actin-based contractile ring divides the cell into two. In cyk-3 mutant embryos, the actin ring fails to divide the cell (midfocus). However, a ring of f-actin is formed at the right time and site, suggesting that cytokinesis is initiated (surface view, arrow).

View larger version (110K): [in a new window]   Fig. 4. Optimized in vitro conditions partially rescue the cytokinesis defect of cyk-3 embryos. (a) In EGM, wild-type blastomeres can divide normally. Cells of the P-lineage divide in a linear cleavage pattern (white arrow); cells of the AB lineage divide in a spiral cleavage pattern (white arrowhead). (b) cyk-3 mutant embryos under the same conditions frequently show cleavage furrows (black arrowhead), indicating that cytokinesis is not a primary defect.

View larger version (81K): [in a new window]   Fig. 5. Comparison of cyk-3 embryos and wild-type embryos under different osmotic conditions. Embryos were dissected into EGM containing the vital dyes FM4-64 and calcein. FM4-64 stains membranes; calcein is a marker for viability (shown in the insets). 0.5x, 1x and 2x EGM refer to the increasing concentration of stock salts in the medium. (a) In hypotonic medium, wild-type and cyk-3 embryos appear swollen, with few cleavage furrows. (b) In 1x EGM, under isotonic conditions, wild-type embryos have normal morphology; they neither shrink nor swell. By contrast, cyk-3 embryos remain swollen, showing no difference from wild-type or cyk-3 embryos observed in 0.5x EGM (compare with a). (c) In hypertonic medium, wild-type embryos respond to the higher ion concentration by shrinking. cyk-3 embryos under the same conditions do not shrink but collapse. The collapsing does not involve membrane invaginations, since the ingression observed at the surface of the embryo does not correspond to membrane staining.

View larger version (60K): [in a new window]   Fig. 6. Map position and protein structure of cyk-3. (a) cyk-3 maps to the central region of chromosome III, between the breakpoint of sDf125 and sma-4. The large genomic locus contains 14 exons coding for a 1175 amino-acid protein with two conserved domains, an N-terminal EF-hand domain and a C-terminal UCH domain consisting of a Cys-Box (UCH1) and a His-box (UCH2). (b) The CYK-3 protein sequence. The two residues marked with black boxes indicate the position of the point mutations leading to premature STOP codons in the alleles t1525 (pos 98) and t1535 (pos 720). The predicted EF-hand domain is underlined in blue; the predicted UCH domain is underlined in red.

View larger version (28K): [in a new window]   Fig. 7. CYK-3 and DOA4 specifically cleave ubiquitin from linear protein fusions. Ubiquitin (UB) and the ubiquitin-like proteins CeSUMO, NEDD-8 and a ubiquitin-like protein encoded by cosmid H06I04 were fused to a truncated version of ß-Gal (ß-Gal) and used as substrates. The fusion proteins were expressed in bacteria together with either CYK-3 or yeast DOA4. Cleavage activity was assessed by probing with an anti-ß-Gal-antibody on a western blot. Both CYK-3 and DOA4 specifically cleave ubiquitin from ß-Gal. In contrast, none of the ubiquitin-like proteins is cleaved from the ß-Gal substrate in the presence of CYK-3 or DOA4.

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