Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
C. elegans body wall muscle is formed after a series of well-orchestrated steps. This thesis describes the plasma membrane dynamics of these migrating embryonic cells and the identification of two genes unc-54/MHB and mem-1 that appear to be involved negatively regulating post-embryonic muscle membrane extensions. During the characterization of embryonic muscle morphogenesis, whereby two rows of cells split and migrate dorsally or ventrally to form the final four muscle quadrants present upon hatching, I observed an anterior migration event, whereby the anterior-most pair of cells in each of the four muscle quadrants extends long processes to the anterior tip of the developing embryo. The anterior-most muscle cells then follow these extensions into their final positions in the developing embryo. Using RNAi and mutant analysis, I have identified laminin as being involved in mediating the dorsal-ventral muscle migrations and that the α-integrin INA-1, the ephrin VAB-2 and its receptor VAB-1 and the Robo receptor SAX-3 indirectly promote the proper extension of the ventral anterior muscle processes by organizing the embryonic neurons so as to provide a clear path for muscle membrane extension. Post embryonically, the loss of either unc-54 (a myosin heavy chain B), or mem-1 (a WD repeat domain protein) results in ectopic membrane extensions from the mature muscle cells. These extensions can be rescued via targeted depletion of actin remodeling or cell adhesion complex components. During the analysis of these mutations I identified a predisposition for generating these ectopic membrane extensions that is conferred by using ectopic expression of PAT-3/β-integrin that is bound to GFP and that using the PAT-3/β-integrin transmembrane domain to localize GFP to the plasma membrane is sufficient to generate this sensitivity.
C. elegans is an excellent model organism for the study of muscle development and maintenance, but we lack a full catalog of genes involved and their specific roles. I have identified and characterized two novel genes, pxl-1 which is essential in C. elegans pharyngeal muscle and cpna-1 which is essential in body wall muscle. PXL-1 is the C. elegans homolog of vertebrate paxillin and contains the four C-terminal LIM domains conserved in paxillin across all species and three of the five LD motifs found in the N- terminal half of most paxillins. PXL-1 antibodies and a full-length GFP translational fusion localize to muscle adhesion sites in the sarcomere, the functional repeat unit in muscle responsible for contraction. In pharyngeal muscle, PXL-1 localizes to ring-shaped structures near the sarcolemma corresponding to podosome-like sites of actin attachment. Loss of paxillin results in lack of pharyngeal contraction, developmental arrest, and lethality. Expression of paxillin solely in the pharynx results in wild type in movement and body wall muscle structure. This demonstrates that in pharyngeal muscle PXL-1 is essential for contraction, whereas in body wall muscle it is dispensable for filament assembly, sarcomere stability, and ultimately movement.CPNA-1 is a copine domain protein essential for myofilament stability and viability in C. elegans. Worms lacking cpna-1 arrest at the two-fold stage of embryogenesis and have disruption of the myofilament lattice. CPNA-1 contains an N- terminal trans-membrane domain, and a copine domain near its C-terminal. Both a GFP translational fusion and antibody specific to CPNA-1 localize to muscle adhesion sites in body wall muscle. CPNA-1 also binds to components of muscle adhesion sites including UNC-89 (obscurin), and the essential muscle protein PAT-6 (actopaxin), which CPNA-1 requires for localization. The essential MYO-3 (heavy chain myosin) protein is initially localized normally in cpna-1 null animals, but becomes mislocalized as contraction begins indicating CPNA-1 is not required for initial assembly of the sarcomere, but is required to maintain structural stability through development. Together, the characterization of PXL-1 and CPNA-1 provide new insight into the organization of muscle adhesion sites in Caenorhabditis elegans.
No abstract available.
Master's Student Supervision (2010 - 2018)
Nearly three billion humans worldwide have helminth infections, which accounts for a global disease burden of 5.2 million disability-adjusted life years. Parasitic nematodes also have a great affect on agriculture, annually destroying 12.3% of global food crops and accounting for an annual loss of approximately $10 billion (USD) worldwide in the sheep and cattle industry. There is widespread resistance to all classes of anthelmintic drugs. The last drug to enter human clinical trials was 35 years ago. There is a great need for new anthelmintics and this concern has been strongly voiced by the World Health Organization and Gates Foundation. Anthelmintics are considered rare, which is why large chemical libraries are a necessity for anthelmintic discovery. I adapted my Caenorhabditis elegans high-throughput technique WormScan to overcome the bottleneck of whole organism screening for anthelmintic discovery. WormScan was used to screen a library of approximately 26,000 molecules. Hits were tested against diverse organisms to identify nematode specific compounds. I took into account preexisting drug resistance of the established anthelmintics. For every class of anthelmintics there is a corresponding C. elegans resistant strain. Screening the hits against these known resistant strains allowed me to determine if any of the compounds target a novel or established anthelmintic pathways. A forward genetic screen and C. elegans genetic tools were employed to identify the mechanism of action of the candidate D19. Molecular modelling was undertaken to elucidate the resistance mechanism and phylogenetic specificity of the D19. I undertook a structure activity relationship to identify structural moieties of D19 activity. The high throughput screen identified 14 new potential anthelmintics, 5 of which had nematode specificity, which indicates low toxic side effects in humans and limited environmental toxicity. Forward genetic screening and molecular modelling identified D19 to be a worm specific mitochondrial complex II inhibitor. The Structure Activity Relationship demonstrated the chemical features needed for D19 activity. C. elegans was used as a surrogate of parasitic nematodes for whole organism anthelmintic discovery. This thesis is a starting point to replenish the pipeline of potential nematicides. Future studies should focus on progressing these hits to a lead status.
To fill a need in the Caenorhabditis elegans community for genomic DNA held in manageably sized clones for complementation assays, a fosmid library was made from the N2 strain. These fosmid clones were aligned to the canonical sequence and cover 80% of the genome, but there were 396 gaps in contiguous coverage spread over the worm’s six chromosomes. In an attempt to fill in some of these gaps in the original fosmid clones’ sequence, we made another library from the Hawaiian geographic isolate CB4856. Our hope was that the divergence, inherent in the deletions containing 517 genes, between the two genomes would aid in the capture of previously gapped regions. This hope was justified. This thesis outlines the production and comparison of the two C. elegans fosmid libraries made from N2 and CB4856 and provides evidence that the way genomic libraries are made can affect the sequences packaged. Combining the two libraries, we now have a total coverage of 92.8% of genes and 90.43% of sequence in relation to the N2 canonical genome.