Gregory C Ellis et al. (2001) International C. elegans Meeting "Characterization of rot-2, a gene required for proper P0 Spindle Orientation"

Gregory C Ellis, Rebecca Lyczak, Bruce Bowerman

[International C. elegans Meeting, 2001]

or362 is a temperature sensitive allele isolated in a screen for embryonic lethal ts mutants. Mutant embryos show defects in centration and rotation of the first mitotic spindle. Using complementation tests and snip-snp mapping, we have found that or362 is an allele to rot-2 , a gene previously identified in a screen for non-conditional maternal effect mutants (Gonczy et al. 1999). In wild-type one-cell stage embryos, shortly following fertilization, the maternal pronucleus migrates from the anterior of the zygote towards the posterior where it meets with the paternal pronucleus. The pronuclei then move back towards the center of the embryo as the centrosome pronuclear complex rotates 90 degrees such that the first mitotic spindle sets up along the longitudinal axis of the one-cell stage embryo called P 0 . The mitotic spindle then moves posteriorly during anaphase, resulting in the anterior blastomere, AB, being larger than the posterior blastomere, P 1 . In rot-2 (or362ts) mutants, the P 0 spindle fails to rotate and remains orientated transversely in the posterior of the embryo. This causes an ectopic cleavage furrow along the a-p axis that bisects the transverse spindle. Furthermore, in rot-2 (or362ts) mutant embryos, the maternal pronucleus frequently fails to migrate posteriorly. In these embryos, the sperm pronucleus-associated centrosomes nevertheless form a transversely oriented spindle. Thus, rot-2 is required for two microtubule-dependent events: migration of the maternal pronuclei, and rotation of the first mitotic spindle. Further studies will focus on the determination of the molecular identity and additional phenotypic characterization of rot-2 . Gonczy et al . (1999). JCB 144 : 927-946.

Gregory C Ellis et al. (2002) European Worm Meeting "Mutational analysis of tbb-2, a beta-tubulin required for proper P0 spindle orientation"

Jennifer Phillips, Gregory C Ellis, Bruce Bowerman, Rebecca Lyczak

[European Worm Meeting, 2002]

In a screen for embryonic lethal temperature sensitive mutants, or 362 was identified which shows defects in centration and rotation of the first mitotic spindle. or362 fails to complement a gene previously known as rot-2 (t1623), which was identified in a screen for non-conditional maternal effect mutants (Gonczy et al. 1999). Using snip-snp mapping, we were able to map and subsequently identify or362 and t1623 as having missense lesions in the GTP-binding and assembly domains, respectively, of one of two embryonically expressed beta-tubulin genes known as tbb-2.

Gregory P Mullen et al. (2001) International C. elegans Meeting "Neurotransmitter transporter genes in C. elegans."

Robert Barstead, Jim Rand, Gary Moulder, Gregory P Mullen

[International C. elegans Meeting, 2001]

With the wealth of genomic sequence data now available, it is possible to begin the analysis of biological phenomena on a genome-wide scale. A particularly intriguing observation is that many genes are members of gene families (sets of genes whose products have closely related structures and/or functions). One such gene family encodes the neurotransmitter transporters and structurally related proteins (the sodium: neurotransmitter symporter family (SNF)). These proteins are required for the efficient clearance of neurotransmitters (and other bioactive molecules) from synaptic clefts. Several of these transporters have well-established roles in behavior and/or neurological disorders. In addition, they are known targets for drugs of psychiatric importance, such as antidepressants, or drugs of abuse, such as cocaine and amphetamines. However, for many, the endogenous substrate and/or cellular functions are unknown. There are at least 18 members of the SNF family in humans and 14 each in D. melanogaster and C. elegans . Sequence comparisons suggest that humans, fruitflies, and nematodes share a core set of SNF proteins, which include the dopamine, serotonin, and GABA transporters. In addition, they each have unique set of SNF proteins that is distinct for each organism. In C. elegans , there are genes that appear to correspond directly to mammalian dopamine ( dat-1 ; see abstract by Duerr et al .), serotonin ( mod-5 ; Ranganathan et al ., 2000) and GABA transporters, as well as a number of orphan transporters (MacGregor et al ., 1997 IWM:377; James et al ., 1999 IWM:436). We now have gene knockouts in five SNF genes (in addition to mod-5 and dat-1 ) and have begun a systematic analysis of their behavioral phenotypes. Thus far, our observations indicate that transporter gene knockouts generally result in mild behavioral phenotypes. We speculate that the mild phenotypes associated with these mutations reflect functional overlaps between genes ('redundancy') or compensatory mechanisms for the degradation/recycling of neurotransmitters.

Gregory Chin et al. (2001) International C. elegans Meeting "Identifying new genes that function in meiotic DNA repair and double-strand break initiation"

Gregory Chin, Anne Villeneuve

[International C. elegans Meeting, 2001]

Meiotic recombination is accomplished by a modified version of the general pathway for repair of double-strand DNA breaks (DSBs). During meiosis, recombination events are initiated by a deliberate induction of DSBs by a conserved topoisomerase-like enzyme, called SPO-11. Repair of these breaks is achieved using core components, such as MRE-11 and RAD-50, that are also crucial for repair of DNA damage induced by exposure to genotoxic insult. These general repair activities work in conjunction with meiosis-specific proteins, some of which function specifically to promote the crossover outcome of initiated recombination events, such as msh-5 and him-14. While a core of widely conserved proteins that play key roles in this process have been identified, for a variety of reasons, it is likely that there are additional important players yet to be discovered. Thus, we are implementing two strategies to identify additional components of the meiotic machinery with particular emphasis on genes that function in DSB formation and/or meiotic DNA repair. The first strategy employs a collection of mutants identified in the lab that are known to be defective in meiotic chromosome segregation and formation of chiasmata (the cytological manifestations of crossovers), but are otherwise largely uncharacterized. Studies from our lab of the conserved recombination genes mre-11 , spo-11 , and msh-5 have provided us with a framework for designing two key assays to identify genes that function either in meiotic DNA repair and/or in DSB formation. The first assay exploits differences in the responses of mutant germ cells to ionizing radiation (IR) as a means of classifying mutants defective at different steps in the recombination process. Mutants that are defective in meiotic DNA repair, such as mre-11 , exhibit both cytologically visible chromosomal abnormalities (reflecting failure of normal repair) and a complete loss of progeny survivorship in response to IR during meiotic prophase. In contrast, for mutants defective only in meiotic double-strand break initiation, such as spo-11 , IR will bypass the defect in recombination initiation resulting in increased chiasma formation and a corresponding increase in progeny survivorship (reflecting improved segregation). These two types of responses are distinct from the response of mutants competent for repair but specifically defective in crossing-over, such as msh-5 , which exhibit neither the improvement in chiasma formation seen in spo-11 nor the meiotic radiation sensitivity seen in mre-11 in response to IR treatment during prophase. A second assay for mutants with possible DNA repair defects is based on our observation of a progressive loss of reproductive capacity in mre-11 mutants. Although mre-11 homozygotes derived from heterozygous parents are fully viable and produce a normal number of embryos (most of which die because of meiotic segregation defects), there is a marked drop both in the number and in the survivorship of embryos produced by succeeding generations. As a result, the strain cannot be propagated as a homozygous stock. This progressive loss of fecundity and viability sets mre-11 mutants apart from many other meiotic recombination-defective mutants and implies that mre-11 plays a role in an essential cellular process. An example of such a role that has been proposed for mre-11 is a role in repairing DSBs that arise as a by-product of replication. Other mutants that cannot be propagated as a homozygous stock may also play a role in an essential DNA repair process. We are currently screening our collection of Him mutants for a DNA-repair-defective or initiation-defective phenotype using these two assays. The second strategy for identifying new meiotic repair proteins involves a collaboration with Simon Boulton and Marc Vidal, who have provided us with a list of candidates shown to interact with MRE-11 in the yeast 2-hybrid system. We are using RNA interference to test whether any of these genes function in meiotic recombination.

Stupp, Gregory et al. (2011) International Worm Meeting "Responses of C. elegans to Pathogenic Challenge."

Stupp, Gregory, Edison, Arthur S., Ajredini, Ramadan, Gulig, Paul A., Robinette, Steven L.

[International Worm Meeting, 2011]

C. elegans releases and responds to many different chemicals necessary for regulating important behaviors such as mating attraction, dauer formation, aggregation, and recognition and differentiation of food and pathogens. The nematode can sense bacterial populations through small-molecule messengers such as acyl-homoserine lactones, and are able to interfere with certain quorum sensing systems. Additionally, C. elegans has a complex olfactory system which allows the nematode to avoid detrimental conditions such as areas of high population density or osmolarity and areas containing pathogenic bacteria. Moreover, this system, which is affected by released pheromones, exhibits behavioral plasticity that allows the nematode to learn and adapt, for example, to avoid an odorant associated with harmful conditions. This study seeks to determine changes in C. elegans behavior and exometabolome, the set of small molecules released into the environment by the worms, in the presence of a pathogen. We collected exudates from a synchronous population of C. elegans under standard conditions and in the presence of synthetic compounds produced by Pseudomonas aeruginosa, for the purpose of identifying worm responses to pathogenic conditions. We acquired 2D NMR spectra of the exudates, and used a novel method developed in our lab for 2D NMR alignment and pattern recognition (Robinette et al., 2011) to identify NMR peaks correlated with worm responses. We also quantified behavioral responses to these pathogen-challenged exudates using custom software that tracks individual nematodes, quantifies reversal frequency and calculates average speed for a set of nematodes on an agar plate in order to identify potential alarm responses. Additionally, we are working on a high-throughput version of the bioassay apparatus that consists of six webcams mounted on a custom lighting unit which will enable recording and analysis of six bioassays simultaneously. The availability of libraries of mutants for both C. elegans and P. aeruginosa makes this an exciting project to probe mechanisms of interspecies chemical interactions. 1. Edison AS. Current opinion in neurobiology. 2009;19(4):378-88. 2. Beale E, et al. Applied and environmental microbiology. 2006;72(7):5135-7. 3. Kaplan F, et al. Journal of chemical ecology. 2009;35(8):878-92. 4. Schulenburg H, Ewbank JJ. Molecular microbiology. 2007;66(3):563-70. 5. Yamada K, et al. Science. 2010;329(5999):1647-1650. 6. Robinette SL, et al. In Press, Analytical Chemistry (2011).

Savill J et al. (2003) Science "Cell biology. Eat me or die."

Gregory C, Savill J, Haslett C

[Science, 2003]

Jennifer B Phillips et al. (2002) West Coast Worm Meeting "Mutations in alpha and beta tubulin genes affect spindle orientation in the one-cell stage C. elegans embryo"

Bruce Bowerman, Gregory C Ellis, Jennifer B Phillips, Rebecca Lyczak

[West Coast Worm Meeting, 2002]

In a screen for embryonic lethal mutations, we isolated several mutations affecting P0 spindle orientation. Two of these, or346ts and or362ts, exhibit severe defects in microtubule dependent events: Pronuclear migration, centration, and rotation of the nucleocentrosomal complex fail in these mutants, resulting in a posterior, transverse P0 spindle. Although polarity markers such as P-granules are properly localized prior to the first division, the abnormal orientation of the spindle and plane of cleavage result in the mis-segregation of P-granules to both daughter cells. Embryos produced by heterozygous or346 hermaphrodites exhibit the same spindle orientation defects as those seen in embryos from homozygous or346ts parents, indicating that this allele is dominant. We mapped or346ts to LG I, between nob-3 and lin-11, placing it very near the -tubulin gene tba-1. The second gene, or362ts, fails to complement a gene previously known as rot-2 (t1673)(Gnczy et al, 1999), and both alleles of this gene were subsequently mapped to the left arm of LGIII in the same region as the -tubulin tbb-2. We sequenced these loci in our mutants and discovered missense mutations in each. Immunofluorescent analysis using antibodies specific to TBB-2 shows that this protein is present at greatly reduced levels in the P0 spindle of tbb-2 (or362ts) embryos. We also obtained a deletion allele from the C. elegans gene knockout consortium, tbb-2 (gk129). tbb-2 (gk129) exhibits temperature-dependent embryonic lethality and mild defects in stability of the P0 spindle. However, pronuclear migration, centration, and rotation are normal, and the strain is homozygous viable, suggesting that tbb-2 function is partially redundant with other -tubulins in the early embryo. As expected, TBB-2 staining is completely absent in tbb-2(gk129) embryos. tba-1 shares 97% homology at the amino acid level with another -tubulin, tba-2. Microinjecting the full-length tba-1 dsRNA into wild-type worms results in a severely reduced mitotic spindle, presumably due to a depletion of multiple -tubulins. To specifically target tba-1, we generated tba-1 3-prime UTR dsRNA. When this is microinjected into wild-type worms, early embryonic divisions are normal and most embryos hatch. However, when 3-prime UTR dsRNA is injected into homozygous tba-1(or346ts)hermaphrodites, pronuclear migration, centration and rotation defects are rescued, resulting in a normal first spindle and restoring viability at the restrictive temperature. We conclude that for both - and -tubulin, the embryonically expressed isotypes are partially redundant in function. Moreover, we find that a partial disruption of these genes is more deleterious to microtubule-dependent processes than the total absence of either protein in the early embryo. We would like to thank Chenggang Lu and Paul Mains for providing antibodies to TBB-2, and the C. elegans Reverse Genetics Core Facility at the University of British Columbia in Vancouver for providing tbb-2 (gk129).

Sengupta P et al. (2014) Traffic "New insights into an old organelle: meeting report on biology of cilia and flagella."

Barr MM, Sengupta P

[Traffic, 2014]

The rising interest of the scientific community in cilia biology was evident from the fact that registration for the third FASEB conference on 'The Biology of Cilia and Flagella' closed out before the early bird deadline. Cilia and flagella are organelles of profound medical importance; defects in their structure or function result in a plethora of human diseases called ciliopathies. 240 clinicians and basic scientists from around the world gathered from 23 June 2013 to 28 June 2013 at Sheraton at the Falls, Niagara Falls, NY to present and discuss their research on this intensely studied subcellular structure. The meeting was organized by Gregory Pazour (University of Massachusetts Medical School), Bradley Yoder (University of Alabama-Birmingham), and Maureen Barr (Rutgers University) and was sponsored by the Federation of American Societies for Experimental Biology (FASEB). Here, we report highlights, points of discussion, and emerging themes from this exciting meeting.

Davis, Gregory M. et al. (2010) Worm Breeder's Gazette "Triton X decreases adherence of C. elegans to pipette tips in liquid medium"

Low, Lloyd, Davis, Gregory M., Boag, Peter R., Sharma, Madhu

[Worm Breeder's Gazette, 2010]

Manoury B et al. (2001) Curr Biol "Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing."

Watts C, Manoury B, Maizels RM, Gregory WF

[Curr Biol, 2001]

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.