MJ Munoz et al. (1999) International C. elegans Meeting "Isolating genes asociated with longevity"

MJ Munoz, DL Riddle

[International C. elegans Meeting, 1999]

Dauer larvae can live for as long as six months, whereas the life span of animals that develop continuosly to the adult is only about two weeks. Temperature-sensitive daf-2 mutants enter the dauer stage constitutively at 25degC, and when grown at permissive temperature their adult life span is doubled (Kenyon et al. 1993). Mutations in daf-16 , encoding a forkhead transcription factor (Ogg et al. 1997, Lin et al. 1997), suppress the increased daf-2 longevity. Thus, expression of some of the genes that are targets of DAF-16 activation may be responsible for increased longevity. We are using the differential display PCR method (Liang and Pardee 1997) to look for differences in gene expression between a daf-2 mutant (long life span) and a daf-2; daf-16 double mutant (wild-type life span). We have identified two differentially expressed genes that have a higher level of expression in the daf-2 mutants. The sequence of these clones predicted two different proteins with unknown function. We have also looked for differences in gene expression between a daf-2; daf-12 double mutant (four times wild-type life span) and a daf-2; daf-12; daf-16 triple mutant (wild-type life span) (Larsen et al. 1995). In this case we have found eleven differentially expressed genes. Seven of them are expressed at higher levels in the triple mutant. We have sequenced two of these and both encode proteins homologous to lectins. The other four genes are expressed at higher levels in the daf-2; daf-12 double mutant. We have cloned and sequenced one, which encodes a protein with unknown function. We are cloning and sequencing the remaining genes. We plan to integrate this information with that obtained from other methods (SAGE analysis, subtraction libraries and microarrays) to select candidates for genes that enhance longevity. We then plan test the effect of their (1) overexpression and (2) loss of function on life span. Kenyon et al. (1993) Nature 366:461-464 Larsen P. et al. (1995) Genetics 139:1567-1583 Liang P. and Pardee, A. B. (1997) Science 257:967-971 Lin K et al. (1997) Science 278:1319-1322 Ogg et al. (1997) Nature 389:994-999

Munoz-Juan A et al. (2023) Chemistry "Exploring the role of metal in the biointeraction of metallacarboranes with C. elegans embryos."

Nuez-Martinez M, Munoz-Juan A, Vinas C, Laromaine A

[Chemistry, 2023]

Cobaltabis(dicarbollides), ferrabis(dicarbollide), and their halogenated derivatives are the most studied metallacarboranes with great medical potential. These versatile compounds and their iodinated derivatives can be used in chemotherapy, radiotherapy, particle therapy, and bioimaging when isotopes are used. These metallacarboranes have been evaluated in vitro and recently in vivo with complex animal models. Lately, these studies have been complemented using the invertebrate Caenorhabditis elegans , a nematode largely used in toxicology. When evaluated at the L4 stage, cobaltabis(dicarbollides), ([o-COSAN]- and [8,8'-I2-o-COSAN]-), exhibited a higher mean lethal dose (LD50) than ferrabis(dicarbollides) ([o-FESAN]- and [8,8'-I2-o-FESAN]-). In this work, we use the C. elegans embryos since they are a complex biological barrier with concentric layers of polysaccharides and proteins that protect them from the environment. We assessed if the metal atom changes their biointeraction with the C. elegans embryos. First, we assessed the effects on embryo development for metallacarboranes and their di-iodinated derivatives. We observed changes in color and in their surface structure. An exhaustive physicochemical characterization was performed to understand better this interaction, revealing a stronger interaction of ferrabis(dicarbollide) compounds with C. elegans embryos than the cobaltabis(dicarbollide) molecules. Unveiling the biological interaction of these compounds is of great interest for their future biomedical applications.

Munoz MJ et al. (1998) Midwest Worm Meeting "ISOLATING GENES INVOLVED IN C.ELEGANS LIFE SPAN."

Riddle DL, Munoz MJ

[Midwest Worm Meeting, 1998]

Dauer larvae can live for as long as six months, whereas the maximum life span of animals that develop continuously to the adult is only about three weeks. Temperature-sensitive daf-2 mutants enter the dauer stage at 25!C even in conditions that are good for growth and reproduction. When the mutants develop at 15!C they do not become dauer larvae, but their adult life span is doubled (Kenyon et al. Nature 366: 461- 464). This effect has been interpreted as indicating that even at the permissive temperature, these nematodes have activated part of a dauer "efficient life maintenance" program. Mutations in daf-16, encoding a fork head transcription factor (Ogg et al. Nature 389: 994-999, Lin et al. Science 278: 1319-1322), suppress the increase in adult life span of the daf-2 mutant. Thus, expression of a particular set of genes (targets of DAF-16 activation) may be responsible for increased longevity. We are using the differential display PCR method to look for differences in gene expression between daf-2 mutants (long life span) and the daf-2;daf-16 double mutant (wild-type life span). Differentially expressed genes will be cloned and tested for the effect of their expression on life span.

Munoz MJ et al. (2000) Midwest Worm Meeting "New mutations that increase life span and thermotolerance."

Riddle DL, Munoz MJ

[Midwest Worm Meeting, 2000]

Adult daf-2 and age-1 mutants exhibit increased longevity and thermotolerance (Lithgow et al. Proc. Natl Acad. Sci. USA 92:7540-44, 1995). To better understand the mechanisms for increased longevity we used resistance to thermal stress as a positive selection to find additional mutants similar to daf-2 and age-1. We isolated a total of 64 thermotolerant animals and 49 of them exhibited a significant increase in life span. Among these 49 mutants 22 were unc-31 alleles, 7 were daf-2 alleles (including two new alleles that are Daf-c only at 27 C) and 5 were age-1 alleles (including one new allele that is like the typical temperature-sensitive Daf-c, forming 100% dauer larvae at 25 C). The other 15 are not daf-2, age-1 or unc-31 alleles and we call them liv (Long Lived and Viable after heat shock). Here we report the phenotypes of liv mutants that define at least six additional genes, including two unc, two Dyf and two non-Dyf mutants that form dauer larvae constitutively at 27 C.

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.

Manuel J Munoz et al. (2002) European Worm Meeting "Positive selection of mutants with increased resistance and longevity."

Manuel A Fidalgo, Donald L Riddle, Manuel J Munoz, Jose M Monje

[European Worm Meeting, 2002]

We developed selective conditions for long-lived mutants by subjecting the first larval stage to thermal stress. At the moment, we have isolated a total of 64 thermotolerant mutants, 50 of them exhibited increased longevity. All the known classes of longevity mutants affected in the dauer signaling pathway were found (Unc, Dyf and Daf-c). 7 of them fail to complement daf-2, 5 age- 1, and 22 unc-31 all of them are known genes involved in the regulation of longevity. We also isolated 2 long-lived alleles of dyf-2 and one of unc-13, those genes were previously identified by other reasons and not related to longevity before. Thirteen additional mutations identified at least four new genes. We call this new mutant class Liv mutants (Long lIved and viable after thermal stress). One of these mutants is liv-6. This mutant resembles the age-1 reference allele, with Daf-c phenotype at 27C, long-lived and similar levels of thermotolerance. age-1 encodes a phosphatidylinositol 3 kinase (PI3K). liv-6 was mapped to the left arm of chromosome I, in the region where the only PI3K adapter subunit worm homologue is located. Sequencing of this gene in the mutant strain and rescue experiments demonstrated that liv-6 encodes for the PI3K adapter subunit.

Munoz MJ (2003) Mech Ageing Dev "Longevity and heat stress regulation in Caenorhabditis elegans."

Munoz MJ

[Mech Ageing Dev, 2003]

Aging is the most complex phenotype for a multicellular organism. This process is now being under severe investigation. Here I will review the different processes known to affect longevity in the nematode Caenorhabditis elegans and their relationship with thermotolerance. All the longevity mutants that have been tested so far show an increase in stress resistance. In particular, long-lived mutants affected in the IGF/insulin pathway and those affected in the germ-line formation are both thermotolerant and long-lived. The mechanisms that activate the stress resistance are now been understood including the DAF-16 fork head transcription factor transport to the nucleus and the activation of genes involved in the defense to stress. The high correlation between stress resistance and longevity suggests that the same molecular activities that defend the cell from stress can defend the cell from the damage caused by aging.

Munoz, N.R. et al. (2017) International Worm Meeting "Understanding sister chromosome movements between sperm meiotic division."

Cota, Vanessa, Wu, Jui-Ching, Chu, Diana, Munoz, N.R.

[International Worm Meeting, 2017]

Mechanisms that segregate chromosomes during male meiosis are poorly characterized. Male meiosis differs from oocyte meiosis in C. elegans in several ways. In sperm, microtubules are organized by centrosomes while oocytes are acentrosomal. Microtubules in sperm do not disassemble as centrosomes split and migrate during anaphase 1 as they do in oocyte meiosis. Microtubules attach to complexes of kinetochores, that include KNL-1, which attaches to chromosomes. KNL-1 localizes around chromosomes in a cup shape and is retained throughout meiosis in sperm, unlike in oocytes where KNL-1 is removed during anaphase. These differences suggest that sperm and oocytes have distinct mechanisms for shifting the orientation of sister chromatids from segregating from the same pole to opposite poles during anaphase 1. It is unknown how microtubule-kinetochore attachment and detachment are regulated to allow sister chromatids to bi-orient to opposite poles when both kinetochores and microtubules do not disassemble. Our goal is to understand how microtubule-kinetochore attachment and detachment are regulated to bi-orient sisters during male meiosis. The retention of KNL-1 during meiosis allows us to study its localization to characterize this chromosomal biorientation. What are KNL-1's movements when sister chromatids change division plane axis' during bi-orientation of spermatogenesis? I hypothesize microtubules use the kinetochore to detach and re-attach to chromosomes to ensure bi-orientation and division plane rotation. Live imaging sperm expressing knl-1::GFP; tbg-1::GFP allows visualization of kinetochore localization and chromosome movement, in reference to the centrosomes, during meiosis. We have been able to characterize the movement of chromosomes as sisters bi-orient, and have observed distinct stages of the process relative to centrosome migration during anaphase 1. It appears that KNL-1 changes localization from a cup shape, absent on one side, to surrounding the chromosomes as sisters change orientation. This suggests as the DNA changes division plane axis, microtubules organized by the centrosomes are actively using kinetochores to change attachments to chromosomes so that they can be bi-oriented correctly. Our data is allowing us insights into mechanisms that allow microtubules to act on chromosomes to segregate them correctly.

Munoz-Jimenez CM et al. (2017) Genetics "An Efficient FLP-based Toolkit for Spatiotemporal Control of Gene Expression in Caenorhabditis elegans."

Askjaer P, de la Cruz Cruz Ruiz P, Torres A, Ayuso C, Dobrzynska A, Munoz-Jimenez CM

[Genetics, 2017]

Site-specific recombinases are potent tools to regulate gene expression. In particular, the Cre and FLP enzymes are widely used to either activate or inactivate genes in a precise spatiotemporal manner. Both recombinases work efficiently in the popular model organism Caenorhabditis elegans but their use in this nematode is still only sporadic. To increase the utility of the FLP system in C. elegans we have generated a series of single-copy transgenic strains that stably express an optimized version of FLP in specific tissues or by heat induction. We show that recombination efficiencies reach 100 percent in several cell types, such as muscles, intestine and serotonin producing neurons. Moreover, we demonstrate that most promoters drive recombination exclusively in the expected tissues. As examples of the potentials of the FLP lines we describe novel tools for induced cell ablation by expression of the PEEL-1 toxin and a versatile FLP-out cassette for generation of GFP-tagged conditional knockout alleles. Together with other recombinase-based reagents created by the C. elegans community this toolkit increases the possibilities for detailed analyses of specific biological processes at developmental stages inside intact animals.

Munoz MJ et al. (2003) Genetics "Positive selection of Caenorhabditis elegans mutants with increased stress resistance and longevity."

Munoz MJ, Riddle DL

[Genetics, 2003]

We developed selective conditions for long-lived mutants of the nematode Caenorbabditis elegans by subjecting the first larval stage (L1) to thermal stress at 30degrees for 7 days. The surviving larvae developed to fertile adults after the temperature was shifted to 15degrees. A total of one million F-2 progeny and a half million F-3 progeny of ethyl-methanesulfonate-mutagenized animals were treated in three separate experiments. Among the 81 putative mutants that recovered and matured to the reproductive adult, 63 retested as thermotolerant and 49 (80%) exhibited a >15% increase in mean life span. All the known classes of dauer formation (Daf) mutant that affect longevity were found, including six new alleles of daf-2, and a unique temperature-sensifive, dauer-constitutive allele of age 1. Alleles of dyf-2 and unc-13 were isolated, and mutants of unc-18, a gene that interacts with unc-13, were also found to be long lived. Thirteen additional mutations define at least four new genes.