Different domains of C. elegans PAR-3 are required at different times in development

AbstractPolarity is a fundamental cellular feature that is critical for generating cell diversity and maintaining organ functions during development. In C. elegans, the one-cell embryo is polarized via asymmetric localization of the PAR proteins, which in turn are required to establish the future anterior–posterior axis of the embryo. PAR-3, a conserved PDZ domain-containing protein, acts with PAR-6 and PKC-3 (atypical protein kinase; aPKC) to regulate cell polarity and junction formation in a variety of cell types. To understand how PAR-3 localizes and functions during C. elegans development, we produced targeted mutations and deletions of conserved domains of PAR-3 and examined the localization and function of the GFP-tagged proteins in C. elegans embryos and larvae. We find that CR1, the PAR-3 self-oligomerization domain, is required for PAR-3 cortical distribution and function only during early embryogenesis and that PDZ2 is required for PAR-3 to accumulate stably at the cell periphery in early embryos and at the apical surface in pharyngeal and intestinal epithelial cells. We also show that phosphorylation at S863 by PKC-3 is not essential in early embryogenesis, but is important in later development. Surprisingly neither PDZ1 nor PDZ3 are essential for localization or function. Our results indicate that the different domains and phosphorylated forms of PAR-3 can have different roles during C. elegans development

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

SummaryFOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity

Regulation of Longevity in Caenorhabditis elegans by Heat Shock Factor and Molecular Chaperones

The correlation between longevity and stress resistance observed in long-lived mutant animals suggests that the ability to sense and respond to environmental challenges could be important for the regulation of life span. We therefore examined the role of heat shock factor (HSF-1), a master transcriptional regulator of stress-inducible gene expression and protein folding homeostasis, in the regulation of longevity. Down-regulation of hsf-1 by RNA interference suppressed longevity of mutants in an insulin-like signaling (ILS) pathway that functions in the nervous system of Caenorhabditis elegans to influence aging. hsf-1 was also required for temperature-induced dauer larvae formation in an ILS mutant. Using tissue-specific expression of wild-type or dominant negative HSF-1, we demonstrated that HSF-1 acts in multiple tissues to regulate longevity. Down-regulation of individual molecular chaperones, transcriptional targets of HSF-1, also decreased longevity of long-lived mutant but not wild-type animals. However, suppression by individual chaperones was to a lesser extent, suggesting an important role for networks of chaperones. The interaction of ILS with HSF-1 could represent an important molecular strategy to couple the regulation of longevity with an ancient genetic switch that governs the ability of cells to sense and respond to stress

Deubiquitylation Machinery Is Required for Embryonic Polarity in <em>Caenorhabditis elegans</em>

<div><p>The <em>Caenorhabditis elegans</em> one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another <em>C. elegans</em> DUB gene, <em>usp-46</em>, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in <em>Schizosaccharomyces pombe</em> are also required in <em>C. elegans</em> raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.</p> </div

Mutation of <i>rpn-10</i> suppresses lethality and polarity defects in <i>math-33(tm3561); usp-47(RNAi)</i> embryos.

<p>(A) Embryonic lethality of <i>math-33(tm3561); usp-47(RNAi)</i> is reduced by either of two <i>rpn-10</i> mutations. Standard error of the mean is indicated by the error bars. <i>n</i>>350 embryos for N2 controls and <i>n</i>>600 for the other genotypes. Asterisks indicate significance compared to <i>math-33(tm3561)</i> single mutants, <i>p</i><0.01, Student's t-test (B) Data showing the suppression of phenotypic defects in early <i>math-33(tm3561);usp-47(RNAi)</i> embryos by <i>rpn-10</i> mutations. (C) Distance in micrometers of the centrosome from the embryo cortex when it is first detectable. <i>rpn-10</i> mutation suppresses the absence-of-pseudocleavage phenotype and the mislocalization of the centrosome compared to <i>math-33(tm3561); usp-47(RNAi)</i> controls. None of the 18 centrosomes observed in column 2 were detached from the paternal pronucleus, indicating that the detachment phenotype was also completely suppressed. Results in column 4 were significantly different in a Student's t-test <i>p</i><0.01 compared to column 1 controls. Data from columns 1, 3, and 4 are also displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003092#pgen-1003092-g005" target="_blank">Figure 5C</a>.</p

<i>usp-46</i> acts redundantly with <i>math-33</i> and <i>usp-47</i>.

<p>(A) Two-cell embryos at interphase (top) illustrate unequal vs. equal first divisions and at P1 mitosis (bottom) show spindle orientations. Maternal genotypes are indicated. Scale bars for each genotype represent 5 µm. (B) Embryonic lethality measured after depleting <i>math-33</i> in <i>usp-46</i> and <i>usp-47</i> mutants. Bars marked with an asterisk are significantly different from the RNAi controls in a t-test, <i>p</i><0.01. Scale bar represents 5 µm.</p

MATH-33 and USP-47 are not enriched at cortex or centrosome.

<p>The left panels are representative images of embryos showing the distribution of GFP::MATH-33 and GFP::USP-47 in one-cell embryos. Both proteins are present in the cytoplasm, but MATH-33 is enriched in nuclei. The right panels are images of embryos showing immunostaining of endogenous MATH-33(red) in the cytoplasm and the nucleus; USP-47(green) primarily in the cytoplasm. Scale bar represents 5 µm.</p

Effect of RNAi/mutant combinations of <i>math-33</i>, <i>usp-46</i>, and <i>usp-47</i> on P1 spindle orientation.

<p>T-T indicates that both AB and P1 cells of two-cell stage embryos divided transversely. The RNAi and mutant combinations of different DUB genes shown here are ordered according to the phenotypic penetrance of the transverse mitotic spindle orientations in P1.</p