Ali, Munira et al. (2021) International Worm Meeting "Effects of a new bacterial pathogen, Bordetella atroposiae, on the genetic fitness of Oschieus tipulae"

Burkeen, Greg, Felix, Marie-Anne, Luallen, Robert, Ali, Munira, Tran, Tuan

[International Worm Meeting, 2021]

Intracellular bacterial pathogens have long been known to cause infectious diseases within the human population. Obligate intracellular pathogens, like Rickettsia parkeri survive and reproduce solely within the host cell, whereas facultative pathogens, like Listeria monocytogenes, are able to exploit multiple host niches. To fully understand the microbial interactions taking place in the host epithelia, we sample for natural pathogens of wild rhabditid nematodes, like Caenorhabditis elegans and Oscheius tipulae. From ecological sampling in Finestere, France, we discovered a new facultative intracellular bacteria inside the intestinal cells of a wild isolate of O. tipulae. Characteristically, this bacterium, which we named Bordetella atroposiae, has a coccobacilli morphology and exhibits a unique filamentation mechanism for intracellular cell-to-cell spreading. With the main purpose of understanding the effects of B. atroposiae on the genetic fitness of the cognate strain, we conducted lifespan and broodsize assays to determine any health and reproductive impacts of the bacterium. We found that B. atroposiae has a severe negative impact on the genetic fitness of the cognate O. tipulae (JU1501) strain. Animals placed on B. atroposiae lived on average for 3 days, compared to worms on OP50 which lived on average for 7 days. Broodsize assays to determine the number of progeny also showed that animals on B. atroposiae produced on average 2 progeny, compared to those grown on OP50 which produced on average 185 progeny. Aside from the cognate strain, we screened a small panel of wild isolates of O. tipulae and found a spectrum of resistance and susceptibility to B. atroposiae. Two strains that are naturally resistant to B. atroposiae were tested as above for fitness effects. We found that the lifespan of these resistant isolates, BA1009, and JU457, were increased compared to the susceptible strains. Collectively, we discovered a new species of facultative intracellular pathogen, B. atroposiae, with high pathogenicity in the cognate and wild-type strains of O. tipulae. We have also found certain wild isolates of O. tipulae have natural resistance to B. atroposiae, indicating some level of coevolution occurring between this bacterium and O. tipulae. In the future, we will map the O. tipulae alleles leading to B. atroposiae resistance.

J Sulcove et al. (1999) International C. elegans Meeting "Development of an Open-ended Muscle Physiology Experiment with C. elegans"

T Allen, J Sulcove

[International C. elegans Meeting, 1999]

Given the broad range of mutant strains that have deficits in behaviors relevant to an undergraduate course in physiology, we sought to develop a discovery-based experiment with C. elegans that would both amplify concepts being learned in class and permit students to participate in the experimental design. The created experiment spans two to three laboratory sessions and requires a minimal amount of equipment. The cyclic interaction of myosin with actin is the basis of tension development in muscle cells, as well as eukaryotic cells generally, and the mechanical events are coupled to the hydrolysis of MgATP by myosin. The experiment challenges students to characterize quantitatively the deficits in muscular function arising from selected missense mutations in unc-54 (myosin heavy chain B). The experiment exploits the availability of unc-54 mutations ( e.g. , s74 and s95 alleles) that have salient functional deficits without disruption of muscle structure. Following discussion of Reynold's numbers, students generally develop swimming assays in which tail-beats per minute are examined as a function of bulk solution viscosity. The concepts that the students consider when designing their experiments include the following: osmolarity and its measurement; bulk solution viscosity and its measurement; muscular fatigue and endurance; and force-velocity relations and their interpretation in the context of muscular efficiency. Students have found the experiment engaging, because of the linkage between mutations in human cardiac myosin and familial hypertrophic cardiomyopathy, a lethal heart disease. Moreover, the accessibility of the crystalline structure of the myosin head (containing the catalytic domain and the actin-binding regions) permits students to correlate their results with the emerging structure-function relationship of myosin. The experiment can also be extended to the analysis of other mutations affecting muscular function ( e.g. , unc-27 /Troponin I-2; please see abstract presented at this meeting by Burkeen et al. , In Vivo Analysis of Troponin I Domains in Muscle Function).

Jennifer B Phillips et al. (2001) International C. elegans Meeting "mir-1 and mir-2 exhibit defects in pronuclear migration and spindle orientation in the P0 stage C. elegans embryo"

Jennifer B Phillips, Rebecca Lyczak, Bruce Bowerman

[International C. elegans Meeting, 2001]

The position of the first mitotic spindle in the C. elegans embryo is crucial for establishing the cellular asymmetry necessary for proper patterning . Events such as pronuclear migration, centration, and rotation of the centrosome/nuclear complex are important precursors to proper spindle orientation, but remarkably little is known about the machinery which enacts these processes. To investigate the genes responsible for these and other early events, we screened for temperature sensitive embryonic lethal mutations with defects in P 0 spindle orientation. We isolated alleles of mel-26, zyg-9, rot-2 (see abstract by Greg Ellis) , and dnc-1 (see abstract by John Willis), in addition to two new loci we have named mir-1 and mir-2 , for pronuclear mi gration and P 0 r otation defective. In mir-1 and 2 mutant embryos, the maternal pronucleus often fails to migrate to the posterior of the embryo before the first mitotic spindle sets up. In these embryos, the maternal DNA may not be integrated into the mitotic apparatus, and instead seems to be randomly segregated to one of the daughter cells, appearing as an extra nucleus. The pronuclei do not centrate in these mutants, and the centrosome/nuclear complex does not rotate, resulting in a posteriorly positioned, transversely oriented spindle and a cleavage plane through the long axis of the embryo. Initial polarity appears normal, but cytoplasmic determinants are subsequently mislocalized due to the aberrant cleavage plane. I am working to characterize these migration and rotation defects in detail, using time lapse video microscopy, immunocytochemistry, and GFP fusion lines in which we can visualize tubulin and histone in living embryos. mir-1 and 2 map to chromosomes I and II, respectively. I am continuing to map each mutant to small intervals on these linkage groups, and will be employing RNAi and transgenic rescue to clone mir-1 and mir-2 .

John Willis et al. (2001) International C. elegans Meeting "The dynactin subunit, dnc-1 (or404ts), is required for mitotic spindle orientation during earlyC. elegans embryogenesis"

Bruce Bowerman, John Willis

[International C. elegans Meeting, 2001]

To understand the mechanisms governing the cell division processes of the early embryo, we have conducted genetic screens to identify temperature sensitive, maternal effect, embryonic lethal mutations. From these screens we have identified a class of mutants with spindle orientation defects at the one-cell stage P0 blastomere (see posters by Jen Phillips, Greg Ellis and Sandra Encalada). One of these mutations, or404ts , was positioned by meiotic mapping near the dnc-1 locus. dnc-1 , the orthologue of the vertebrate gene p150Glued, is a component of the multi-subunit dynactin complex, which is required for all known functions of the microtubule-associated dynein motor protein. Sequence analysis of dnc-1 from or404ts animals identified a missense mutation in the C terminus of the protein. To our knowledge, or404ts is the only known mutant allele of dnc-1 in C.elegans. Roles for dynein and dynactin in the early embryo were revealed by studies in which known components, including dnc-1 , were inhibited by RNAi (1,2). These studies show that dynein and dynactin are required for multiple microtubule dependent events in the early embryo; these include pronuclear migration, centrosome migration, and rotation of the centrosomal/nuclear complex. In wild type embryos, the sperm contributed centrosome duplicate and migrate to opposite poles of the male pronucleus, transverse to the anterior-posterior (a-p) axis of the embryo. Meanwhile, the female pronucleus migrates to meet the male pronucleus at the posterior of the embryo. This centrosomal/nuclear complex then rotates 900 to lie along the polarized a-p axis of the embryo before the completion of the cell cycle. In dnc-1 ( or404ts ) mutant embryos pronuclear migration and centrosome migration occur as in wild-type, but centrosomal/nuclear complex rotation does not occur, resulting in a mitotic spindle which aligns transverse to the a-p axis. Since dynein and dynactin RNAi mutant embryos exhibit early defects in embryogenesis, a role for dynein and dynactin in spindle orientation past the one cell stage has not been addressed. In both the P1 blastomere and the EMS blastomere at the 2 and 4 cell stage, respectively, the mitotic spindle rotates to align along the a-p axis. It is not known if dynein or dynactin are required for these rotations. To address this we are performing temperature up-shift experiments with dnc-1 ( or404ts ) to determine its role in these rotations. Preliminary experiments show that P1 spindle orientation is aberrant when dnc-1 ( or404ts ) embryos are shifted to the restrictive temperature after a wild-type first division (1/3 embryos). This suggests that dynactin and dynein may function in P1 spindle orientation. Further characterization of this function and a possible requirement for dnc-1 ( or404ts ) in EMS spindle orientation will be covered in this poster. 1. Skop A. and White J. (1998) Current Biology 8: 1110-1116. 2. Gonczy et al., (1999) JCB,14(1) : 135-150.