Waaijers, Selma et al. (2011) International Worm Meeting "Tissue-specific identification of polarity protein complexes."

Heck, Albert, Munoz, Javier, Boxem, Mike, Waaijers, Selma

[International Worm Meeting, 2011]

The polarization of epithelial cells depends on the activity of three main polarity complexes: the Crumbs, PAR-3, and Scribble complexes. The domain architecture of these proteins suggests that they function as scaffolding proteins, recruiting and interacting with multiple protein partners. We are attempting to systematically identify novel interaction partners for these proteins through an affinity purification/mass spectrometry (AP/MS) approach. AP/MS approaches to date have mainly been used in unicellular systems, such as yeast or tissue culture. In multicellular organisms such as C. elegans, the composition of protein complexes likely varies between tissues. For example, depending on the tissue type examined, PAR-3 is found in a complex with PAR-6 and aPKC, or PAR-3 and PAR-6/aPKC seem to have independent functions (e.g., in C. elegans epidermal epithelial cells or the Drosophila blastoderm epithelium). To accurately determine the composition of protein complexes, it is necessary therefore to develop technologies to purify proteins from specific tissues. In addition, this would allow us to compare protein complexes between different tissues, potentially revealing functional insights. To purify proteins from specific tissues we are using an in vivo biotinylation based approach. Proteins of interest are tagged with GFP and a small (14aa) peptide (the Avi tag), that can be recognized and biotinylated by the bacterial biotin ligase BirA. To tag the proteins, we are using a fosmid-based recombineering approach, which allows us to maintain all the regulatory sequences controlling the expression of the protein. Transgenic lines expressing the GFP-Avi tagged protein are crossed with lines expressing BirA from tissue-specific promoters, resulting in biotinylation of the tagged protein only in the tissue of interest. Protein complexes are then purified from worm lysates using streptavidin beads. To identify the components of these complexes, we are using a quantitative mass spectrometry approach.

Apfeld J et al. (1996) West Coast Worm Meeting "A GENETIC SCREEN FOR LONG-LIVED WORMS."

Hsin H, Dorman JB, Kenyon CJ, Apfeld J, Albinder B

[West Coast Worm Meeting, 1996]

We are interested in finding genes that control lifespan in C. elegans. We are currently doing a clonal screen for mutations that lead to an increase in lifespan. We mutagenize a temperature-sensitive sterile strain, clone out F2 progeny, allow these worms to self fertilize, clone some of their progeny into a new plate at the restrictive temperature, and screen these for increased lifespan. So far we have screened 360 F2 clones and are in the process of retesting 3 of them. Javier Apfeld is also studying the gene daf-2. The e1370 allele of this gene has multiple phenotypes: at 25°C daf-2(e1370) worms develop into dauer larvae (a developmentally arrested, stress resistant, long lived alternative larval form); at 20°C daf-2(e1370) worms do not form dauer larvae, but live 2.5 times longer than wild-type (N2) worms. Javier wants to know where daf-2 functions and how it acts, and is currently doing mosaic analysis to address these questions.

Galli, Matilde et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A quantitative mass spec approach reveals phosphorylation of the spindle positioning protein LIN-5 by the anterior PAR complex"

Heck, Albert, Galli, Matilde, Munoz, Javier, Van den Heuvel, Sander

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During asymmetric cell division, a cell first acquires an axis of polarity, and then aligns the mitotic spindle along this polarity axis to generate daughter cells with different fates. Although it is becoming clear how polarity cues are established, it is unknown how these cues regulate the position of the mitotic spindle to direct the plane of division. The conserved LIN-5/GPR/G? complex is thought to act downstream of polarity signals to generate microtubule pulling forces, but a direct link between polarity proteins and the LIN-5/GPR/G? complex has not been identified. To determine whether this complex is directly controlled by polarity proteins, either in protein composition or by post-translational modifications, we set up a quantitative mass spectrometry approach. For this, early C. elegans embryos were differentially labeled with either 15N or 14N isotopes and treated with either control RNAi or RNAi against the polarity protein kinase pkc-3. LIN-5 immunoprecipitations from the 14N/15N mixed lysates were then analyzed by mass spectrometry. Strikingly, depletion of pkc-3 resulted in a six-fold reduction in phosphorylation of four residues on LIN-5, while all other phosphorylation sites were unaltered. Thus, PKC-3 promotes the phosphorylation of LIN-5 in early embryonic divisions. Using in vitro kinase assays with recombinant LIN-5 we found that PKC-3 directly phosphorylates these residues on LIN-5. To determine the role of these phosphorylations during spindle positioning, we are currently analyzing non-phosphorylatable and phospho-mimicking LIN-5 mutants. Preliminary results indicate that LIN-5 phospho-mimicking mutants have reduced spindle pulling forces and have defects in asymmetric cell division. Together, these results suggest that PKC-3 controls spindle positioning through direct phosphorylation of LIN-5.

Galli, Matilde et al. (2011) International Worm Meeting "PKC-3 aPKC phosphorylates LIN-5 NuMA to position the mitotic spindle during asymmetric division."

Portegijs, Vincent, Galli, Matilde, Munoz, Javier, Van den Heuvel, Sander, Heck, Albert, Boxem, Mike

[International Worm Meeting, 2011]

Asymmetric cell divisions that create different daughter cells are critical for development and stem cell maintenance. The position of the mitotic spindle controls the plane of cell cleavage and determines if polarized cells divide symmetrically or asymmetrically. Studies in various animal systems have identified an evolutionarily conserved PAR-3/PAR-6/aPKC polarity complex that acts upstream of a conserved LIN-5 (NuMA)/GPR (Pins/LGN)/Ga pathway to position the mitotic spindle. However, the molecular interactions between polarity proteins and LIN-5/GPR/Ga remain to be identified. We found that the C. elegans LIN-5 protein is phosphorylated in vivo by the polarity kinase PKC-3 (aPKC), and that this phosphorylation directs the position of the mitotic spindle during asymmetric division. We developed a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to C. elegans embryos, we found that depletion of the polarity kinase PKC-3 results in severely reduced phosphorylation of four adjoining LIN-5 serine residues. These LIN-5 residues are also phosphorylated by PKC-3 in vitro. LIN-5 phosphorylation in vivo overlaps with PKC-3 localization at the anterior cell cortex and temporarily coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase anterior-directed spindle movement, while phosphomimetic mutations reduce spindle migration. Together, these results demonstrate that PKC-3 locally inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5, thereby promoting posterior migration of the spindle. These results reveal an important molecular mechanism by which polarity cues control the spindle-positioning machinery to instruct the cleavage plane during asymmetric cell division.

The, Inge et al. (2013) International Worm Meeting "Critical targets of CYD-1/CDK-4 in the control of cell cycle entry."

Prinsen, Martine, Heck, Albert, Van den Heuvel, Sander, Munoz, Javier, Ruijtenberg, Suzan, The, Inge

[International Worm Meeting, 2013]

Whether to continue or to arrest cell proliferation is a key developmental decision. Ultimately, this decision depends on the activity of cyclin-dependent kinases (CDKs) in the G1 phase of the cell cycle. G1 cyclin/CDKs counteract inhibition of cell-cycle entry by the retinoblastoma (Rb) tumor suppressor protein. It is currently unknown if additional critical regulators of G1 progression are regulated by CDK phosphorylation. We have previously shown that C. elegans has single cyd-1 Cyclin D and cdk-4 Cdk4/6 genes that are essential for G1 progression. Inactivation of lin-35, the sole C. elegans Rb family member, partly overcomes CYD-1/CDK-4 requirement. However, cell proliferation remains limited in double mutants of lin-35 and cyd-1, or lin-35 and cdk-4, indicating the presence of critical CYD-1/CDK-4 target(s) in addition to the LIN-35 protein. To identify such targets, we performed a genetic screen for mutations that restore cell proliferation in the absence of cyd-1 and lin-35 function. The screen identified a single mutant with normal development and cell proliferation patterns. We found that the responsible mutation results in incomplete loss-of-function of fzr-1 Cdh1, a substrate specificity factor of the anaphase promoting complex (APC/C). The APC/CCdh1 E3 ubiquitin ligase targets proteins for degradation and is critical for exit from mitosis and inhibition of cell-cycle entry in G1. Using in vitro kinase assays, we found that CYD-1/CDK-4 phosphorylates LIN-35 on specific sites that correspond to Rb-inactivating phosphorylation in mammalian cells. In addition, CYD-1/CDK-4 phosphorylated eight different serine residues in the N-terminal region of FZR-1 Cdh1. In other systems, phosphorylation of the Cdh1 N-terminus by mitotic CDKs prevents APC/C association and premature APC/CCdh1 activation. We propose that similar phosphorylation by CYD-1/CDK-4 overcomes APC/CCdh1 activity in G1, thereby promoting cell cycle entry. Our data support that the LIN-35 Rb and FZR-1 Cdh1 proteins are the only two critical phosphorylation targets of Cyclin D/CDK-4 in the control of cell-cycle entry.

M Mercedes Perez-Jimenez et al. (2008) European Worm Meeting "Characterization and identification of the long-lived mutant liv-7(pv17)"

Jose M Monje, M Mercedes Perez-Jimenez, Manuel J Munoz

[European Worm Meeting, 2008]

Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.. Using a positive selection for thermotolerant mutants (Munoz et al.. Genetics, 2003. 163: 171-80) we have isolated liv-7(pv17), a long-lived. mutant that does not show any visible phenotype.. We have done a double mutant with the temperature sensitive allele daf-2. (e1370)III, which is also long-lived and encodes to the only insulin. receptor homologue that exists in C. elegans. The double daf-2 (e1370);. liv-7 (pv17), shows a phenotype different to any of the single mutants, an. L1 arrest at 25degrees. Besides this phenotype we noticed that liv-7 (pv17). enhances other phenotype characteristics of daf-2 mutants in different. alleles like e1370, m577 or e979.. The longevity of the mutants of the Insulin/IGF pathway depends of the. transcription factor daf-16, in the same way the longevity of liv-7 (pv17). is suppressed by daf-16. This result allows us to think that liv-7 is a. negative regulator of the transcription factor daf-16.. We are now actively working in the mapping of liv-7 for its molecular. identification.

Manuel J Munoz et al. (2005) International Worm Meeting "Characterization of the long-lived mutants liv-7 and liv-8"

Ana M Brokate-Llanos, Manuel J Munoz, Isabel Calmarza-Font, Jose M Monje

[International Worm Meeting, 2005]

Using a positive selection for thermotolerant mutants (described in Munoz et al. Genetics 2003 163:171-80) we have isolated a total of 78 survivors of the thermal stress selection, among them, 24 are long lived. The characterization of two of those mutants will be described here. Mutation in the liv-7 gene produces a 40<sym05>/o increase of longevity, this effect is dependent of the fork head transcription factor daf-16. Beside the longevity phenotype, liv-7, at 27C, shows a weak delay in development, a phenotype that is independent of daf-16. Using the delay in development phenotype as surrogate genetic marker, we have located liv-7 in a small interval of the chromosome X, where no other long-lived mutant has been identified, suggesting that liv-7 encodes for a new gene involved on regulation of ageing. liv-8 shows a more pleiotropic phenotype. At 16C shows severe migration defects and at 25C it is lethal, when eggs are incubated at 25C majority of the embryos die, and those that do not die, stop development before reaching adulthood. However, if they are shifted up from 16C to 25C when adults, they are long lived. Mapping and further characterization of those mutants are on going.

Lyons LC et al. (1997) Worm Breeder's Gazette "Acute Ethanol Exposure Induces Nondisjunction of the X Chromosome During Spermatogenesis"

Hecht RM, Lyons LC

[Worm Breeder's Gazette, 1997]

In Drosophila, short term and chronic exposure of young females to ethanol causes an approximately 10 fold rise in chromosome nondisjunction (Rey et al., 1992). However, the question remains as to whether this effect is caused by ethanol or its primary metabolite, acetaldehyde (Rey et al., 1994). Caenorhabditis elegans provides a good model for studying this effect because X chromosome nondisjunction can be easily monitored by determination of the number of male progeny produced following acute chemical exposure. Ethanol, in optimal experimental conditions of 30 minute exposures of mid to late L4s to 7% ethanol in M9 salts, caused a greater than 10 fold increase in the production of male progeny (13.2 males/1000 worms compared to 1.03 males/1000 worms for M9 alone with approximately 10,000 F1 progeny counted for each class). Concentrations as low as 5% generated a five fold increase in nondisjunction than controls while 10% and 12.5% ethanol concentrations also resulted in a 10 fold increase in nondisjunction. Higher concentrations of ethanol proved too toxic as noted by egg laying defects and reduced brood sizes. Synchronous populations of N2 worms grown at 20 degrees were tested at different developmental stages (according to age and criteria described by Kimble and White, 1981) to determine the most sensitive stage to 30 minute exposures of 10% ethanol in M9 salts. When L1s, L2s and L3s were exposed to ethanol, only the normal background numbers of males were observed. Early L4s treated with ethanol showed a slight rise in number of males while mid to late L4s treated with ethanol produced significantly more males than the controls. However, young adults as well as older adults showed no increase in the number of male progeny when treated with ethanol. The stage most sensitive to short-term ethanol exposure correlates with the time when the majority of sperm are produced. This suggests that ethanol induces meiotic nondisjunction during spermatogenesis. Surprisingly, acute exposures of mid to late L4s to acetaldehyde concentrations ranging from 0.1% to 1.0% did not produce a higher rate of chromosome nondisjunction compared with M9 controls, approximately 12,600 F1 progeny of 0.1% acetaldehyde treated L4s were counted producing 1.11 males/1000 worms. Hermaphrodites treated with 0.5% acetaldehyde had similar numbers of male progeny while 1.0% treated hermaphrodites had such small brood sizes that no male progeny were observed. This study suggests that ethanol in Caenorhabditis elegans causes chromosome nondisjunction rather than its primary metabolite, acetaldehyde. Kimble, J.E. and White, J.G. (1981). On the Control of Germ Cell Development in Caenorhabditis elegans. Dev. Bio. 81, 208-219. Rey, M., Palermo, A.M. and Munoz, E.R. (1992). Nondisjunction induced by ethanol in Drosophila melanogaster females. Mutation Research. 268, 95-104. Rey, M., Palermo, A.M. and Munoz, E.R. (1994). Lack of effect of acute acetaldehyde treatment on X chromosome segregation in Drosophila melanogaster females. Mutation Research. 320, 1-7.

Ana Maria Brokate-Llanos et al. (2006) European Worm Meeting "Characterization of the Long-Lived Mutant liv-8(pv18)"

Ana Maria Brokate-Llanos, Manuel J. Munoz, Jose M. Monje

[European Worm Meeting, 2006]

Characterization of the Long-Lived Mutant liv-8(pv18). Using a positive selection for thermotolerant mutants (Munoz et al. Genetics 2003 163: 171-80) we have isolated liv-8(pv18), a thermotolerant and long-lived mutant. The phenotype of liv-8(pv18) is very pleiotropic, at 16C shows severe gonad migration defects and when incubated at 25C they are embryonic lethal.. Because thermotolerance it has been also observed in mutants related to the dauer formation pathway, we have studied the interactions with mutants of the TGF-B and the insulin/IGF dauer signalling pathways. At 20C liv-8(pv18) enhances the dauer formation phenotype of the TGF-B mutants daf-1 and daf-7 but does not affect this feature in mutants of the insulin/IGF pathway. The insulin/IGF pathway is involved also in regulation of longevity. daf-2(e1370) mutants increase wild-type life span, and the double with liv-8(pv18) lives even longer, suggesting that liv-8(pv18) may be involved in a different pathway to daf-2 regulating longevity. This is also supported by the evidence that, differently to daf-2 mutants, liv-8(pv18) increases longevity without relocating the transcription factor daf-16 into the nucleus. The interaction with daf-16 is interesting because we showed that this gene is needed for the increase of longevity of liv-8(pv18).. Mapping analysis indicate that is located on the right arm of the chromosome I, actually we are actively working in its identification.

Ayuso, Cristina et al. (2019) MicroPubl Biol

Ayuso, Cristina, Askjaer, Peter

[MicroPubl Biol, 2019]

Several techniques are available for spatiotemporal control of genome recombination and gene expression in the nematode Caenorhabditis elegans. Here we report a novel tool to combine the powerful FLP-Frt and GAL4-UAS systems to increase their versality and to offer additional levels of control.FLP is an enzyme that catalyzes recombination between two short Frt DNA sequences and is frequently used to excise genomic fragments flanked by Frt sites, thereby either activating or knocking out gene expression, depending on the experimental design (Hubbard, 2014). Recently, we generated multiple strains that stably express FLP in different somatic tissues from single-copy transgenes and demonstrated that they in most cases induce recombination in ~100% of the cells of the expected tissue (Munoz-Jimenez et al., 2017). We subsequently constructed a strain for germline recombination to permanently knock out Frt-flanked genes or exons (Macas-Len and Askjaer, 2018).The GAL4-UAS system is based on the Saccharomyces cerevisiae Gal4p transcription factor and its cognate DNA target called upstream activating sequence (UAS). Typically, this bipartite system includes a series of driver strains expressing GAL4 in specific tissues and one or several strains with an effector gene downstream of UAS repeats. Wang and colleagues from the Sternberg laboratory recently optimized the GAL4-UAS system for C. elegans (cGAL) and reported several tissue-specific cGAL drivers (Wang et al., 2017). Moreover, they have developed a split cGAL toolkit where the DNA binding and activation domains are expressed as individual polypeptides, thereby enabling further fine-tuning of spatiotemporal control: only when and where the two components are co-expressed they will activate the UAS::effector transgene (Wang et al., 2018).