Yen, C. et al. (2017) International Worm Meeting "Mitochondrial ALH-6 is essential for sperm quality and regulates male reproductive senescence."

Curran, S.P., Lynn, D.A., Yen, C.

[International Worm Meeting, 2017]

Reproduction is essential to perpetuate life. Mitochondria integrity and functionality has been linked to proper sperm function across multiple species. Most studies have examined the negative impact of the environment or acute stress plays on sperm function and reproductive output. However, the mechanistic impact that normal cellular metabolism plays in the regulation of sperm quality and activity remains unclear. Here, we show that C. elegans with mutations in alh-6, a conserved proline metabolism gene, display early reproductive senescence. Loss of proline catabolism results in specific deficits in sperm number, size, and activation. These defects in sperm quality are linked to changes in mitochondria morphology, metabolic output, and reactive oxygen species (ROS) generation. Intriguingly, the reproductive defects in alh-6 mutants are not simply due to reduced flux through the proline catabolism pathway. Instead the premature reproductive senescence in alh-6 mutants is caused by aberrant ROS homeostasis and loss of energy storing metabolites; however the relative impact that altered levels of ROS and metabolic intermediates plays in the sperm number, size, and activation phenotypes is remarkably different. Finally, the expression of the mammalian ortholog of alh-6, Aldh4a1, is significantly reduced with age in mouse testes suggesting a potential conserved role of alh-6 in male reproductive fitness. Taken together, we have uncovered a novel role for a conserved and central amino acid catabolism pathway on normal sperm function and our work uncovers a new variable to measure which can predict and alter the rate of aging of the male reproductive system.

HA Tissenbaum et al. (1999) International C. elegans Meeting "Using model organisms to identify genes that affect life span"

L Guarente, HA Tissenbaum

[International C. elegans Meeting, 1999]

Previous work in C. elegans has identified a number of mutants that affect life span. We are trying to identify new components of the aging mechanism by isolating mutations in genes that are conserved in C. elegans and have previously been shown be important in the aging process of other eukaryotic organisms. 1) Werner Syndrome : People with Werner Syndrome (WRN) show many signs of premature aging (reviewed in Yu et al. 1997). The WRN protein is a member of the RecQ family of helicases. In yeast, mutations in the WRN homolog, SGS1 , cause yeast to age prematurely (Sinclair et al. 1997). Four members of the RecQ family have been identified in the C. elegans database. We are currently isolating knockout mutations of all four of the family members. As well, we are generating GFP fusions and doing RNAi to help elucidate the different functions of these four genes. These studies will help to determine if any of the four genes is the true WRN homolog in C. elegans and whether they function to control aging. 2) SIR2 homologs: Studies from yeast ( S. cerevisiae ) have identified the SIR proteins (SIR2, 3, 4) as important components for yeast aging (reviewed in Sinclair et al. 1998). SIR2, however, is unique among the SIR proteins in several respects including that it is highly conserved from yeast to man. There are two SIR2 homologs identified in the C. elegans database. To understand the role of C. elegans SIR2 in aging, we are generating knockout mutations and GFP fusions as well as performing RNAi. We are also using the candidate gene approach to identify mutants in the SIR2 homologs. References: Sinclair D.A., Mills K., and Guarente L. (1997). Science 277 :1313-6 Sinclair D.A., Mills K., and Guarente L. (1998). Annu Rev Microbiol 52 :533-60 Yu C.E., Oshima J., Wijsman E.M., et al. (1997) Am J Hum Genet 60 :330-41

Zhang H et al. (1996) East Coast Worm Meeting "Role of Cell Signalling in C. elegans Male Ray Development"

Zhang H, Emmons SW

[East Coast Worm Meeting, 1996]

Cell-cell interactions play an important role in C. elegans male ray development, but the nature of the signals involved is unknown. The presence of cell signalling has been demonstrated by cell ablation experiments. For example, if either of the seam cells V5 or V6, which give rise respectively to ray1 and to rays 2-6, is killed, its anterior neighbor cell will adopt the fate of the killed cell. An interaction between seam cell T, which generates rays 7-9, and its anterior neighbor, V6, can be demonstrated in a pal-1 mutant background. pal-1 is a homeobox transcription factor that is the C. elegans homolog of Drosophila caudal (1). In pal-1 homozygotes, V6 makes no rays; however, if the T cell is killed, V6 generates a normal complement of rays (2). Mosaic analysis has shown that the action of pal-1 to promote rays in the V6 lineage is due to its action within that lineage (1). This suggests that a ray-inhibiting signal normally passes from T to V6, but is blocked or overridden by the action of pal-1. The target of pal-1 may be the homeobox gene mab-5, since presumptive activation of mab-5 in a mab-5 gain-of-function mutant results in the generation of V6 rays in a pal-1 mutant background (1). In order to identify the signal from T that inhibits ray generation by V6 and to identify possible components of a mab-5 regulatory circuit, we are isolating extragenic suppressors of pal-1. We have obtained 19 alleles from about 4000 genomes screened. Some are very strong (more than 90% of V6 lineages make rays); others are weak (about 30% of V6 lineages make rays). In addition to resulting in recovery of V6 rays, most of these mutants have other phenotypes, such as ray fusion and abnormal ray morphology. Mapping and phenotypic characterization of these new mutations is in progress. 1 Waring, D.A & Kenyon, C Nature 350:712-715 2 Waring, D.A & Kenyon, C Cell 60:123-131

Guo S et al. (1995) International C. elegans Meeting "PAR-1: Asymmetrically Localized Kinase Required for Embryonic Polarity"

Kemphues KJ, Guo S

[International C. elegans Meeting, 1995]

The par-1 gene is required for many aspects of embryonic polarity including unequal cleavage and the asymmetric distribution of cytoplasmic components. We cloned the par-1 gene using a combination of YAC transformation rescue and antisense phenocopy. It encodes a putative serine/threonine kinase with strong similarity to kinases identified in yeasts and mammals. Two par-1 alleles have mutations in invariant kinase residues, suggesting that the kinase activity is crucial for PAR-1 function. PAR-1 protein is localized to the posterior periphery of the zygote. After the first embryonic division, PAR-1 is detected at the P1 periphery but is absent from the AB periphery. As the cell cycle proceeds, the peripheral PAR-1 becomes asymmetrically localized and partitioned into P2. Asymmetric localization of PAR-1 occurs again in P2 and P3, but does not occur in P4. The localization of PAR-1 shows a striking correlation with the distribution of P granules, and thus raises the possibility that PAR-1 is not only required for the early anterior-posterior polarity, but is also involved in later P lineage asymmetric divisions or germ-line development. To better understand interactions among par genes, we examined PAR-1 distribution in other par mutants. Interestingly, in par-2 mutants, PAR-1 is not detectable at the periphery of the zygote or P lineage blastomeres, but in par-3 mutants and in par-2 par-3 double mutants PAR-1 is present around the entire periphery of all cells in the early embryo. These results, together with other results from our lab (see abstracts by B. Etemad-Mogadam and Lynn Boyd), lead us to propose that PAR-1 is restricted to the posterior by PAR-3 activity and that PAR-2 is required to properly localize PAR-3.

Zappaterra MD et al. (2000) West Coast Worm Meeting "Loss of a dynamin related protein MGM-1 causes excessive mitochondrial fragmentation"

van der Bliek A, Zappaterra MD

[West Coast Worm Meeting, 2000]

Our lab studies the functions of dynamin and dynamin related proteins. These proteins form a small family of large GTP-binding proteins. We have recently shown that the C. elegans dynamin related protein DRP-1, is involved in the scission of the mitochondrial outer membrane (1). Since little is known about mitochondrial division and mitochondrial morphology in animal cells, we are investigating the mechanisms that regulate these processes. MGM-1 is another member of the dynamin family present in animal cells. As of now we know that this protein is present in yeast, C. elegans, and humans. Unlike other members of the dynamin family, MGM-1 has an N-terminal mitochondrial leader sequence. MGM-1 is also more closely related to bacterial dynamin-like proteins than it is to dynamin or DRP-1, suggesting that this protein has followed a different evolutionary path. We speculate that MGM-1 was introduced into eukaryotic cells by the progenitors of mitochondria. Expression studies using -galactosidase under the control of the mgm-1 promoter show high levels of expression in intestines, body wall muscles, and neurons. These high levels might reflect high metabolic rates in those tissues, because the MGM-1 expression pattern is similar to that of another dynamin-related protein, DRP-1, which is also important for mitochondrial maintenance. Mgm-1 RNAi drastically slows the growth rate and approximately doubles the life span of C. elegans. We show that excessive fragmentation of mitochondria is induced by RNAi and mgm-1 antisense cDNA. We present evidence that MGM-1 is localized to the mitochondrial matrix where it might regulate the morphology of the mitochondrial inner membrane. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular Cell 4: 815-826. 1999.

Lynn Boyd (2001) International C. elegans Meeting "A simple lab using single worm PCR and dpy-5"

Lynn Boyd

[International C. elegans Meeting, 2001]

A simple lab has been designed for use in a sophomore genetics course. There are four primary objectives for this laboratory exercise: 1) students predict genotypes based on phenotype 2) students design primers for PCR 3) students use the PCR technique 4) students analyze results using gel electrophoresis The learning elements of the lab can be broken down into two main areas. First, it serves as a demonstration of the relationship between genotype and phenotype. Students observe the phenotype of worms and then make predictions about the genotype which they subsequently test. A second valuable learning element has been having the students design primers for PCR. Students must have a good understanding of both DNA structure and DNA polymerase activity to design primers successfully. This laboratory exercise makes use of a dpy-5 mutation that contains a deletion of 1009 bp (gracious thanks to Colin Thacker and Ann Rose for supplying the strain).. The mutation (e907) is semidominant. Therefore, students can identify all three genotypes in a population of worms containing the wild type and e907 alleles (+/+; +/e907; e907/e907). The investigation occurs over three lab periods. In the first lab, students become familiar with observing the Dpy phenotype and with picking worms. They also design primer pairs that will amplify both the wild type and e907 alleles. In the second lab, students use these primer pairs in single worm PCR reactions. They are asked to pick worms that represent each of the three genotypes and use these for PCR. In the third period, an agarose gel is run of the PCR reactions. Students determine the genotypes of the worms they chose and find out if their genotype predictions were correct. I have found that sophomore students are able to do this lab with a high percentage of success. If you would like more information or would like the lab handout, please contact Lynn Boyd at boydl@uah.edu.

Narayan, Anusha et al. (2009) International Worm Meeting "Transfer at a thermosensory synapse in C. elegans."

Laurent, Gilles, Sternberg, Paul, Narayan, Anusha

[International Worm Meeting, 2009]

C.elegans is an attractive system for neural circuit analysis. To analyze the functional dynamics of circuits that control behavior, it is necessary to understand the underlying synaptic transformations. Thermotaxis is an established behavior in C. elegans[1-2]. We attempt to characterize the transfer function at a prominent synapse within the thermotactic circuit: between AFD, the primary thermosensory neuron[3], and AIY, its principal post-synaptic partner. We drive expression of Channelrhodopsin-2(chR2), a light-activated cation channel [4], solely in AFD, using a cell-specific promoter, and use whole-cell patch-clamp recording techniques to measure the light-evoked synaptic response at AIY. We are able to reliably activate the presynaptic cell AFD, evoking depolarizing potentials of up to 40 mV and inward currents of up to 15 pA. The postsynaptic response at AIY is small: less than 5 mV, with inward currents of less than 1 pA, and is graded and tonic, lasting the duration of the stimulus. The response reverses around 0 mV and seems to be frequency independent, showing no obvious short-term facilitation or depression. Our results further validate the use of chR2 to stimulate neural activity, and indicate that this synapse has low gain, and transmits information from AFD to AIY with short latencies and high fidelity. It will be interesting to see how AIY integrates this information with other incoming streams, and to examine processing downstream in the thermotactic circuit. 1.Mori, I., H. Sasakura, and A. Kuhara, Worm thermotaxis: a model system for analyzing thermosensation and neural plasticity. Curr Opin Neurobiol, 2007. 17(6): p. 712-9. 2.Ryu, W.S. and A.D. Samuel, Thermotaxis in Caenorhabditis elegans analyzed by measuring responses to defined Thermal stimuli. J Neurosci, 2002. 22(13): p. 5727-33. 3.Clark, D.A., et al., The AFD sensory neurons encode multiple functions underlying thermotactic behavior in Caenorhabditis elegans. J Neurosci, 2006. 26(28): p. 7444-51. 4.Boyden, E.S., et al., Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci, 2005. 8(9): p. 1263-8.

Tauffenberger A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Translation frameshiftings as a new mechanism of toxicity for the expanded CAG diseases"

Parker A, Tauffenberger A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The expansion of CAG trinucleotides coding for glutamine (polyGln) is the cause of at least nine late-onset neurodegenerative diseases. The expanded CAG (expCAG) diseases may have some pathogenic mechanisms in common as the disease threshold for all of these disorders is around 40 CAG repeats and they display misfolded intracellular protein aggregation phenotypes. We have turned to the model organism C. elegans to explore mechanisms of expCAG toxicity Translational errors may be a major cause of disrupted protein homeostasis1, and mistranslation may be relevant to genes with expCAG tracts since frameshift errors to ([GCA]n) also produce expanded polyAlanine (polyAla). Large polyAla tracts are missing in the proteomes of almost all species compared to polyGln. The sequenced genome of H. sapiens revealed a low percentage of polyAla-containing genes (0.80%)2. Few proteins have 10-15 Ala residues and none with greater than 20 in the three species examined2. Experimental evidence suggests that expanded polyAla are intrinsically more toxic than polyGln3. Thus, over time mistranslation may be particularly harmful for the expCAG diseases since they result in polyGln/polyAla hybrids that may be more toxic than polyGln alone. Indeed, -1 frameshifting/mistranslation has been proposed as a novel mechanism of expCAG toxicity4. We are creating transgenic frameshift reporter strains expressing full-length ATXN3 protein, involved in Spinocerebellar Ataxia 3, the most common type of ataxia to investigate this phenomenon. Our model and a summary of our recent findings will be presented. 1.Drummond, D.A. & Wilke, C.O. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell 134, 341-52 (2008). 2.Lavoie, H. et al. Polymorphism, shared functions and convergent evolution of genes with sequences coding for polyalanine domains. Hum Mol Genet 12, 2967-79 (2003). 3.Latouche, M. et al. Polyglutamine and polyalanine expansions in ataxin7 result in different types of aggregation and levels of toxicity. Mol Cell Neurosci 31, 438-45 (2006). .Gaspar, C. et al. CAG tract of MJD-1 may be prone to frameshifts causing polyalanine accumulation. Hum Mol Genet 9, 1957-66 (2000).

Driscoll MA et al. (2000) East Coast Worm Meeting "Contributions of WRN-related Helicases to C. elegans Aging"

Driscoll MA, Herndon LA, Nycz KM

[East Coast Worm Meeting, 2000]

Four genes in the C. elegans genome exhibit sequence similarity to the RecQ family of DNA helicases which include the human Werner (WRN), Bloom (BLM) and Rothmund-Thomson (RTS) syndrome genes. Mutation of human WRN and RTS can confer features of accelerated aging. Both WRN and RTS patients have a high rate of genome instability, which is also a key feature of patients with Bloom syndrome. Similarly, mutation of the yeast RecQ homolog, SGS1, results in a high rate of genomic instability and a lifespan 40% shorter than wild type1, 2. To determine if the RecQ family of helicases share similar functions in C. elegans, we generated deletions within the C. elegans gene most closely related to the Werners gene (F18C5.2) and in another homolog (E03A3.2) and have tested these strains for lifespan and genomic instability. Preliminary results indicate that animals lacking F18C5.2 or E03A3.2 are viable and fertile. They have normal lifespans and do not have a high rate of genomic instability. Since C. elegans has four related helicases, there may be redundancy in the activity of these helicases. To test this hypothesis, we are now analyzing animals that carry mutations in multiple DNA helicase family members. It has recently been determined that him-6 mutant animals, which have a high rate of chromosome nondisjunction, contain mutations in the C. elegans DNA helicase gene T04A11.6, which is most closely related to the human BLM gene3. We are also testing mutants for UV radiation or X-ray hypersensitivity, since it is possible that these helicases play a role in DNA repair. We are testing the expression pattern of these genes and are screening for a deletion in the fourth remaining C. elegans DNA helicase family member, K02F3.1. By establishing a nematode model for accelerated aging disorders, we hope to extend understanding of the action of helicase family members in senescence and learn more about mechanisms of aging overall. Watt, P.M., I.D. Hickson, R.H. Borts and E.J. Louis. 1996. Genetics 144: 935. Sinclair, D.A., K. Mills and L. Guarente. 1997. Science 277: 1313. Wicky, C., A. Rose and F. Mueller. 1998. European Worm Meeting Abstract.

Ayako Hasegawa et al. (2003) International Worm Meeting "C.elegans homologue of yeast Mdm38 and mammalian Letm1 is essential for mitochondrial morphology"

Ayako Hasegawa, Alexander M van der Bliek

[International Worm Meeting, 2003]

Mitochondria are dynamic structures that divide and fuse throughout the life of a cell. Several proteins have been identified to affect mitochondrial outer membrane division and fusion. Our lab discovered that dynamin related protein called DRP-1 plays a key role in mitochondrial outer membrane division (1). The detailed analysis of the dominant-negative phenotype of DRP-1, which causes a block in outer membrane division, indicates that inner membrane division occurs independent of outer membrane division. 3D reconstruction of mitochondrial inner membrane and cristae by electron tomography also indicates that fusion occurs within mitochondria. Cristae often fuse to each other or to inner membrane (2). These data suggest that there are separate mechanisms of division and fusion for inner membrane aside from those for outer membrane, and a mechanism that coordinates different membrane remodelling. Our lab has been looking for novel proteins that are involved in regulation of mitochondrial morphology. C.elegans body wall muscle cells are used because they are large and flat, and their mitochondria are fairly well organized. Mitochondrially targeted fluorescence protein marker under muscle-specific promoter was used to visualize the mitochondria with minimum background. We are analyzing the function of a C.elegans homologue of the yeast Mdm38 protein, which has previously been implicated in mitochondrial morphology (3). This protein is also homologous to the mammalian protein Letm1, which is deleted in some patients with Wolf Hirschhorn Syndrome, a disease of as yet unknown etiology. We find that this protein is localized to mitochondria and affect mitochondrial morphology. Strong MDM38 loss-of-function causes abnormally enlarged and distended mitochondria, while mild loss-of-function causes enlarged and indented mitochondria, resembling oxidation-phophorylation complex loss-of-function phenotypes. Over expression of MDM38 appears to cause loss of mitochondrial potential and shriveling of matrix and/or swelling of outer membrane. We suspect that MDM38 homologous protein affects cristae remodeling, however the functionality of this protein is yet to be discovered. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular, Cell 2: 815-826. 1999. 2. Frey, T.G., Mannella, C.A. Trends Biochem Sci 25(7): 319-324. 2000. 3. Dimmer, K.S., Fritz, S., Fuchs, F., Messerschmitt, M., Weinbach, N., Neupert, W., Westermann, B. Mol Biol Cell 13(3): 847-853. 2002.