Nearly all multi-cellular organisms have mechanisms that allow specific tissues to withstand substantial mechanical stress without rupture or permanent deformation. These mechanisms often involve structures composed of extracellular matrix (ECM) proteins (e.g. elastin and fibrillin) that assemble into elastic fibers or elastic fiber-like structures that can stretch and flex but return to their original shape once force is removed. Information obtained from studies of elastic polymers found in nature has numerous potential applications in materials science, medicine and military projects (e.g. in tissue grafts, protective clothing and energy-absorbing soundproofing). The PI and co-workers have discovered novel elastic fiber-like structures in the nematode C. elegans that connect pharynx and adjacent muscle cells, and have named these structures nematode elastic fibers (NEFs). As the animals forage for food, NEFs stretch, bend, flex and pivot and help to position the pharynx in the center of the body cavity. In previous work, Dr. Vogel has identified two essential components of the NEF: the highly conserved ECM proteins hemicentin and fibulin-1D. These proteins co-assemble at NEFs and two other cell junctions that are also flexible and resistant to mechanical stress. The specific aims of the current proposal utilize the power of the C. elegans genetic system to: 1) dissect the mechanism of hemicentin and fibulin-1D assembly into NEFs and 2) determine the composition of these intriguing elastic polymers by identifying the other molecular NEF components. The long-term goals are to define the essential requirements for assembling NEFs, to assemble NEFs or NEF-like structures in vitro and a detailed analysis of the mechanical/biophysical properties of these intriguing structures.
The intellectual merit lies in the potential to provide detailed information into the assembly and composition of a novel type of cell junction. Although cell-cell and cell-ECM junctions have been the focus of intense investigation, hemicentin and fibulin-1D are unique in that they always assemble in a sandwich at junctions between cells (cell-ECM-cell) where they form a novel type of elastic and flexible biological glue.
There are two aspects of broader impacts that pertain to this project. One is the potential for societal benefit through the likelihood that the work will have relevance for materials science and bioengineering of synthetic biocompatible elastic materials that would be useful in a variety of applications. In addition to the potential utility of the research itself, another broader impact of the proposed research will be to utilize C. elegans as an ideal model organism to teach basic concepts in biology. In conjunction with the experimental goals of the research, molecular biology and genetics learning modules will be developed for UMBIs established, state-wide science education program. This program consists of three components that serve Maryland K-12 students: 1) UMBIs on-site teaching laboratory 2) Maryland Loaner Lab (MDLL) in which the necessary equipment and consumables are sent to teachers so they can enhance their lab offerings and integrate new curriculum in their classrooms and 3) a 32-station mobile teaching laboratory housed in a tractor trailer that travels throughput the state of Maryland to provide hands-on laboratory education for K-12 students. An additional benefit is that separate teacher professional development programs are available through all three components designed to provide teachers with an opportunity to expand their laboratory skills and enhance the content of their bioscience instruction.
Intellectual Merit: Nearly all multi-cellular organisms have elastic fibers or elastic fiber-like structures that enable tissues to resist significant amounts of mechanical stress without rupture or permanent deformation. Studies of elastic fibers found in nature are likely to provide insights into the design of materials with desirable biophysical properties for military, biomedical and industrial use. The goals of this project were to investigate the assembly, structure and function of elastic fiber-like structures in the nematode C. elegans. Previous work by our laboratory demonstrated that the primary components of these elastic structures are hemicentin and fibulin-1D, two evolutionarily conserved secreted components of the extracellular matrix. A series of molecular genetic constructs were designed to determine the regions of fibulin-1 and hemicentin required for their co-assembly. Expression of the proposed constructs in C. elegans resulted in several key insights into the domains within fibulin-1D required for elastic fiber assembly including the fact that the second EGF repeat is essential for blocking ectopic interaction with hemicentin and that this activity may be protease dependent. This insight is important because it demonstrates that elastic fiber assembly is highly regulated and that this regulation prevents elastic fibers from assembling in inappropriate places. A manuscript describing these findings has been recently published (Muriel et al., 2012). The second focus of the funded work was on the function of elastic fibers. A role for hemicentin based elastic fibers in the cleavage furrow during germ cell cytokinesis in C. elegans was identified: In the absence of hemicentin, germ cell cleavage furrows are unstable and frequently retract, resulting in cytokinesis failure. Remarkably, we also found that loss of hemicentin-1 in mouse embryos results in cleavage furrow retraction and cytokinesis failure. These results have been published (Xu and Vogel, 2011) in a paper selected as ‘Editor’s choice in Developmental Biology’ in The Scientist (May 2011). More recently, we have extended these findings, providing substantial evidence for a model whereby mutations in hemicentin that result in cytokinesis failure produce a tetraploid intermediate cell with a multipolar spindle that may continue to divide (Vogel et al., 2011). This model provides an explanation for the isolation of hemicentin mutants in 1979 in a screen for C. elegans genes that are required for correct germline chromosome segregation and produce a "him" (high incidence of male progeny) phenotype when mutated (Hodgkin et al., 1979). Finally, in follow-up studies, we have identified a transmembrane protein that appears to be essential for stability of the cleavage furrow in dividing cells. This transmembrane protein is found in cytoplasmic vesicles that traffic to the cleavage furrow as cytokinesis begins. Exocytosis of cytoplasmic vesicles results in the deposition of phospholipid that contributes to cleavage furrow growth. However, our data suggests that secreted and transmembrane proteins that are among cytoplasmic vesicle contents make critical contributions to cleavage furrow growth, structure and stability. Broader Imapct: The PI actively participates in multiple Bioscience Education and Outreach Programs. For example, Dr. Vogel routinely hosts trainees that include high school students and undergraduates who do not have access to state of the art technology. In addition to a variety of skills necessary for C. elegans research (e.g. basic microscopy, microinjection, transgenesis, mutant screens) the trainees receive training in the fundamentals of experimental design, data analysis and critical thinking. Recent trainees include high school students Jasmine Johnson, Joseph Hunter, and Meghan Loughery and high school biology teachers Stephanie Hammond, Hannah Jardine, and Allan McDonald, who were participants in the NSF-funded (DUE-0831970) ExPERT (Extended Professional Experiences in Research to Teachers) Program, Jahnavi Patel, Eboselumhen Iseghohi, and Sarah Kramb, undergraduate students from Stevenson University, Towson University, and University of Maryland College Park, respectively. Dr. Vogel was a speaker and participant in a two day Curriculum Development Workshop designed to bring together Maryland middle and high school teachers with University of Maryland higher education faculty and education and outreach staff. The Workshop was based on Project Lead the Way’s (www.pltw.org/curriculum-philosophy/curriculum-philosphy.cfm) successful curriculum development program that brings in a diverse group of teachers to brainstorm about what students should know based on Maryland State Department of Education’s Voluntary State Curriculum and Core Learning Goals. Dr. Vogel presented his research to a group of 10 high school science teachers and then worked with the group to develop an outline for a classroom activities based on his study organism, C. elegans. The workshop was a great success and the teachers reported enjoying learning about Dr. Vogel’s research and came away with many new ideas for their classroom.
