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"Cell and process migration are critical to the establishment of neural circuitry. The study of these processes is facilitated through use of model organisms with simple nervous systems, such as C. elegans. Research in this nematode has defined the cytoplasmic adaptor MIG-10 as a key regulator of these processes. Mutation of mig-10 disrupts neuronal and axonal migration and outgrowth of the ‘canals’, or processes, of the excretory cell. MIG-10 directs the localization of UNC-34, which remodels actin filaments at the leading edge of a migrating cell or process to modify the direction or rate of its protrusion. An interactor of MIG-10 identified in a yeast two- hybrid analysis, ABI-1, has several roles in actin remodeling, such as targeting Ena/VASP members for phosphorylation by Abl kinase. Mutation of abi-1 in the nematode produces phenotypes that resemble those of mig-10 mutants, including disrupted outgrowth of the excretory canals, a developmental process in which ABI-1 is known to function cell autonomously. To test the hypothesis that the ABI-1/MIG-10 interaction contributes to cell migration and outgrowth, both in vivo and in vitro analyses were performed. Expression of either MIG-10A or MIG-10B exclusively in the excretory cell partially rescued the canal truncation characteristic of mig-10 mutants, suggesting MIG-10 functions autonomously in this cell during canal outgrowth. Physical interaction between MIG-10 and ABI-1 was confirmed using a co-immunoprecipitation system. Both MIG-10A and MIG-10B interact with ABI-1 through a mechanism that likely involves the SH3 domain of ABI-1 and sites in either the central region or C-terminus of MIG-10. These results suggest that MIG-10 and ABI-1 function together in a cell autonomous manner to promote cell or process migration. A possible consequence of this interaction is modulation of the MIG-10 binding to UNC-34 through Abl-mediated phosphorylation of MIG-10."

"August 2004" / Bibliography: leaves 220-255. / xiii, 255 leaves, [22 p.] : ill., plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The author has investigated the formation, and protein compostition, of the outer dense fibres and fibrous sheath of a model marsupial species, the brush-tail possum (Trichosurus vulpecula). / Thesis (Ph.D.)--University of Adelaide, Dept. of Anatomical Sciences, 2004

We have previously reported that non-muscle myosin II (NMMII) and myosin light chain kinase (MLCK) are required for oncogenic Ras signaling to ERK in Ki-Ras transformed rat fibroblsasts (Helfman and Pawlak, J. Cell Biochem. 95(5), 1069-80, 2005). Here I examine if MLCK plays a role in ERK signaling in various tumor derived human epithelial cell lines. I also determined whether genetic inhibition of NMMII isoforms IIA and IIB, or MLCK also inhibits ERK activation in the MCF 10A human breast epithelial cell line expressing oncogenic H-Ras. Inhibition of MLCK by pharmacological inhibitors such as ML-7 and ML-9 was used to determine the role of MLCK in ERK signaling in an array of H/K-Ras transformed and tumor derived cell lines: T-24 bladder carcinoma, HCT 116 colon carcinoma, and MCF 10A Ras breast cancer cells. Genetic inhibition was carried out using specific siRNA targeted towards MLCK and NMMIIA or IIB. The knock down of NMM IIA and IIB did not inhibit active ERK, which suggested either a redundant function of NMM IIC or an alternate substrate for MLCK. Inhibition of MLCK by ML-7/ML-9 reduced activated ERK in all H/K-Ras transformed, or human tumor derived cell lines we tested. The possible mechanism of how MLCK could play a role in ERK signaling was tested by co-immunoprecipitation (co-IP) of MAPK scaffolding proteins with MLCK. That the ERK scaffold KSR1 regulates ERK signaling in MCF 10A Ras, was demonstrated through inhibition of KSR1 with siRNA. Moreover, KSR was shown to interact with MLCK because it was found to co-precipitate with MLCK.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

The potential of determining stem cell fate through mechanoregulation has been demonstrated recently. However, the underlying mechanism remains largely unknown. Previously, we developed a novel microencapsulation technique to entrap cells in a nanofibrous collagen meshwork and use the cell-collagen model to study mechanoregulation of human mesenchymal stem cells (hMSCs). Initially, hMSCs were randomly distributed within the construct. Upon cyclic compression, hMSCs reoriented towards a direction along the loading axis. Cytoskeleton, being the major sub-cellular machinery supporting cell shape and motility, should play crucial role in sensing and responding mechanical signals. Therefore, a better understanding in the change of cytoskeleton and associated molecules upon mechanical loading is a prerequisite to rationalizing the loading regimes for stem cell-based functional tissue engineering. In the current project, we hypothesize that hMSCs encapsulated in 3D collagen construct will respond to cyclic compression by remodeling the cytoskeleton structures and altering the interactions with collagen matrix. hMSCs collagen construct were cyclically compressed for 9 hours through micromanipulator based compression system. After compression, constructs were harvested either immediately after compression, 2 hours after compression and 24 hours compression, together with non-loading control group. Here, we report compression-induced novel changes in cytoskeleton. Firstly, omnidirectional filopodia-like structures together with stress fibers bucking were observed immediately after 9hrs of cyclic compression. Secondly, actin patches were observed shortly after removal of 9hrs compression before the actin fibers resumed. Apart from exhibiting similar morphology with filopodia, the omnidirectional filopodia-like structures may share a similar function in interacting with ECM. Co-localization of the major membrane-bound matrix metalloproteinases MT1-MMP with actin staining was found along the length of the filopodia-like structures. A local collagen digestion zone, characterized by the presence of collagenase cleaved collage, was found co-localizing at least partially with the filopodia-like structures around the cell. Whether creating pericellular collagen digestion zone was mediated by MT1-MMP along the compression-induced filopodia like structures and what functions the digestion zone serves are interesting question to answer in the future. Another interesting observation is the complete disassembly of pre-existing stress fibers followed by formation by numerous actin patches throughout the cell shortly after removal of the compression loading. Stress fibers reformed in 24 hours after removal of the loading. Quantitative measurement of F:G actin ratio agrees with such disassembly and reassembly dynamics. Colocalization of actin branching protein arp2/3 with the actin patches was found, suggesting that mechanically loaded hMSCs were re-establishing actin cytoskeleton network from these nucleation centers. Further studies are required to figure out the underlying significance of the loading-induced cytoskeleton dynamics in hMSCs and whether the actin patches Arp2/3 complex associates with endocytosis of cleaved collagen fragments. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

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