Browsing by Subject "actin"
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Item Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle(2004-12-01) Shepard, Athena A.; Julian Borejdo; Thomas Burghardt; Ben HarrisShepard, Athena A., Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle. Doctor of Philosophy (Molecular Biology and Immunology), December, 2004, 161 pp., 1 table, 42 illustrations, bibliography, 253 titles. Muscle contraction results from the interaction of myosin and actin proteins contained in the muscle sarcomere. During actomyosin interactions, myosin consumes ATP and imparts an impulsive force to actin resulting in sliding of myosin and actin filaments to produce work. These proteins constitute the elementary motor responsible for cellular motility. The overall goal of this research project was to elucidate the mechanism of the actomyosin interaction on a molecular level. Novel time-resolved optical microscopic techniques followed myosin and actin orientation changes during skeletal muscle contraction. Fluorescence anisotropy was used to study the real time orientation changes of myosin, actin, and nucleotide during a single cross bridge cycle beginning in a state of rigor. Rabbit psoas fibers were isolated on a microscopic slide and labeled with fluorescently labeled regulatory light chain to monitor orientation changes of the lever arm of myosin, with fluorescent phalloidin to monitor orientation changes of actin and/or with Alexa ADP to monitor ATP hydrolysis. Caged ATP was perfused into the fiber prior to analysis to allow a small population of cross-bridges to execute a single cross-bridge cycle. Flash photolysis with UV light during analysis converted caged ATP from an inactive from to an active from. Confocal and multi-photon imaging allowed illumination of a small population of fluorescently labeled cross-bridges to measure orientation changes over time. The conclusions of this dissertation are: 1) The regulatory light chain rotates during skeletal muscle contraction and the lever arm model is supported, 2) Release of ADP from S1 corresponds to a single rotation of the lever arm, 3) Actin rotates during skeletal muscle contraction, 4) The rotation of actin is passive, i.e. it rotates as a consequence of dissociation of S1 from actin. The results revealed orientation changes in key contractile proteins during muscle contraction in the non-disease state organism. By understanding the mechanism of muscle contraction in the healthy scenario, hopefully a better understanding of diseased states stemming from mutations in contractile proteins (Usher’s Syndrome, Snell’s Waltzer Disease, and certain familial hypertrophic cardiomyopathies) will be made available, leading to a better preventative measures or treatments to treat such diseases in the future.Item MIEN1 Drives Breast Cancer Invasion by Regulating Cytoskeletal-Focal Adhesions Dynamics(2015-05-01) Kpetemey, Marilyne F.; Vishwanatha, Jamboor K.; Clark, Abbot F.; Basu, AlakanandaIn the recent years, Migration and Invasion Enhancer 1(MIEN1) has emerged as a potential biomarker and a plausible target in breast cancer. Located in the 17q12-21 region of the human chromosome, next to the Her-2/neu loci, MIEN1 presents a robust expression in breast carcinomas; however is completely absent or low in the normal tissues. MIEN1 is post-translationally modified by geranyl-geranyl transferase-I (GgtaseI), which adds isoprenyl group to the carboxyl-terminal of the protein. Prenylated MIEN1 then associates with the inner leaflet of the plasma membrane and acts as an adaptor protein triggering downstream signaling through the Akt/NF-kB axis to regulate the expression of key proteases and angiogenic factors like MMP-9, uPA and VEGF. In migrating cells, MIEN1 enhances filopodium formation at the leading edge. Aside from its prenylation and redox-active motifs, MIEN1 also contains a canonical ITAM, reported to be associated with epithelial-to-mesenchymal transition. Although the role MIEN1 in cell migration and invasion is well known, the underlying molecular mechanisms remain elusive. Here, we show that MIEN1 interacts with Annexin A2, a cytoskeletal protein and a regulator of the plasminogen/plasmin system in breast cancer cells to increase migration and invasion. We confirmed that MIEN1 regulates actin dynamics by associating with cytoskeletal effectors in the lamellum. We also show that MIEN1 expression redirects breast tumor cell migration toward a collective migration. Our studies validate MIEN1-ITAM and CAAX as key motifs to MIEN1-induced functions. In conclusion, our findings confirm the role of MIEN1 in the remodeling of the actin cytoskeleton during motility. Furthermore it attests to previous findings suggesting that motility patterns depend on various environmental factors along with regulatory genes involved. Our study demonstrates an interesting example from cell biology where adaptor proteins regulate various signaling pathways and control cellular processes through protein-protein interactions.