In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2009/11/30). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 134-144). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 134-144). Also available on the Internet.

This study investigated spatial learning in Caenorabditis elegans; the ability to associate reinforcing cues with a location. Naive, wildtype C. elegans were trained in a microfluidic t-maze in the presence of diacetyl (a volatile attractant associated with food) and subsequently tested to see if they could associate diacetyl with one arm of the t-maze. 70-80% of the subjects chemotaxed towards diacetyl during training phase, but they randomly chose left or right when diacetyl was absent (number of subjects that chose diacetyl being under 65%). From our experiments, it is unlikely that the worms are associating diacetyl with one arm of the t-maze, but appears to be using some component of the atmosphere as a cue.

Aging is a complex process by which a combination of environmental, genetic and stochastic factors generate whole-system changes that modify organ and tissue function and alter physiological processes. Over the last few decades, many genetic and environmental modulators of aging have been found to be highly conserved between humans and a diverse group of model organisms. Yet, an integrative understanding of how these environmental and genetic variables interact over time in a whole organism to modulate the systemic changes involved in aging is lacking. The goal of this thesis project is to advance a systems perspective of aging by providing the experimental tools and conceptual framework for dissecting the regulatory connection between environmental inputs, molecular outputs and long term aging phenotypes in Caenorhabditis elegans, an experimentally tractable multi-cellular model for aging. Specifically, this work advances the quantitative imaging toolsets available to biologists by developing and refining microfluidic, hardware, computer vision, and software integration tools for high-throughput, high-content imaging of C. elegans. As a result of these technological advances, new roles for the TGF-beta and serotonin signaling pathways in encoding environmental food signals to influence longevity were uncovered and quantitatively characterized. Moreover, this work develops and integrates new microfluidic technologies with off-chip support systems to establish a platform for long-term tracking of the health and longevity trajectories of large numbers of individual C. elegans. The capabilities of this platform have the potential to address many important questions in aging including addressing environmental determinants of aging, the sources of inter-individual variability, the time course of aging-related declines and the effects of interventional strategies to improve health outcomes. Together, the toolsets for quantitative imaging and the long-term culture platform permit the large-scale investigation of both the internal state and long-term behavioral and health outputs of an important multicellular model organism for aging.

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Studying animal mechanics is crucial in order to understand how signals in the neuromuscular system contribute to an organism’s behaviour and how force-sensing organs and sensory neurons interact. In particular, the connection between the nerves and the muscles responsible for the force generation in the neuromuscular system needs to be established. Knowledge of the locomotion forces can be beneficial for the development of therapies for muscle disorders, neurodegenerative and human genetic diseases, such as muscular dystrophy. The simplicity of the nematode Caenorhabditis elegans’ (C. elegans) nervous system, which is limited to 302 neurons, has made it an excellent model organism for studying animal mechanics which include mechanosensation and locomotion at the neuronal level. The advent of miniaturized force sensing devices has led to the proposal of various approaches for measuring C. elegans locomotion forces. However, these existing devices are relatively complex, involving complicated microfabrication procedures and are incapable of measuring forces exerted by C. elegans in motion. This thesis addresses these shortcomings by introducing a force sensor capable of continuously measuring the forces generated by C. elegans in motion. The system consists of a micropillar-based device made of polydimethylsiloxane (PDMS) only and a vision-based algorithm for resolving the worm force from the deflection of the cantilever-like pillars. The measured force is horizontal and equivalent to a point force acting at half of the pillar height. The microdevice, sub-pixel resolution for visual tracking of the deflection, and experimental technique form an integrated system for measuring dynamic forces of moving C. elegans with force resolution of 3.13 uN for worm body width of 100 um. A simple device fabrication process based on soft-lithography and a basic experimental setup, which only requires a stereo microscope with off-the-shelf digital camera mean that this method is accessible to most biological science laboratories. The results demonstrate that the proposed device is capable of quantifying multipoint forces of moving C. elegans rather than single-point forces for a worm sample. This allows one to simultaneously collect force data from up to eight measurements points on different worm body parts. This is a significant step forward as it enables researchers to explicitly quantify the relative difference in forces exerted by the worm’s different body segments during the worms’ movements. The device’s capability to determine multipoint forces during nematode motion can also generate meaningful data to compare forces associated with different worm body muscles, gaining new understanding on how these muscles function. The forces measured during locomotion in the micropillars could also be used to differentiate mutant phenotypes. Apart from locomotion forces, the device is also capable of conducting concurrent measurement of other locomotion parameters such as speed, body amplitude and wavelength, as well as undulation frequency. This additional information can be useful to further quantify phenotypic behaviour of C. elegans and deepen the understanding of the theory behind worm locomotion forces. The relationship between C. elegans locomotion forces and their environment has also been analyzed by variation of the pillar arrangement and spacing. The results indicate that the microstructured environment significantly affects the worm’s contraction force, locomotion speed and the undulation frequency. In addition, an alternative measurement technique was provided to measure worm forces on other substrates, such that worm locomotion behaviour in varying environments can be investigated further. The combination of the conventional measurement technique with the findings of worm locomotion on a glass substrate reported show promise for biological measurements and other sensing application such as tactile force. Additional functions of on-chip worm selection, sorting, and imaging have also been integrated with the device, rendering its potential to accommodate for high-throughput application of C. elegans force measurement and locomotion studies in the future. The primary contributions of this thesis are centered around four topics: the development of the PDMS micropillar array and its application to study C. elegans locomotion forces, the analysis of C. elegans muscular forces and locomotion patterns in microstructured environments, the investigation of the worm locomotion forces using different substrates and finally the integration of the PDMS micropillar with PDMS microvalve for on-chip worm selection and imaging. Although the results presented in this thesis focus on wild type C. elegans, the method can be easily applied to its mutants and other organisms.

No description available.