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Double labeling of myosin and actin in migrating NIH3T3 fibroblast |
Overview of Research Interests
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Double labeling of myosin and actin in migrating NIH3T3 fibroblast |
Movement is a fundamental characteristic of living things. The ability of cells to move is critical for normal embryogenesis and tissue formation, wound healing, and defense against infection; it also plays an important role in disease processes such as tumor metastasis. In addition, movement of cellular components within cells is necessary for chromosome separation during mitosis, hormone secretion, phagocytosis, and endocytosis. These cellular and intracellular movements are powered by molecular motors that move along actin-based microfilaments (myosin) and tubulin-based microtubules (dynein). At the tissue and organism level, the contraction of muscle, maintenance of blood pressure, and beating of the heart are also powered by these motor molecules. Mutations in one of these motor molecules, cardiac myosin, are responsible for an inherited heart disease called hypertrophic cardiomyopathy--a common cause of sudden death among otherwise healthy adults.
Our goal is to understand how these motors are regulated and how they convert chemical energy into mechanical force; to define the extent of their involvement in intracellular, cellular, and tissue function and their contribution to disease; and to ultimately begin to develop therapies for the treatment of disease caused by defects in these molecular motors. To address these questions, we have employed a wide array of experimental systems and approaches:
The single-cell amoeba Dictyostelium discoideum serves an extremely powerful genetic tool to rapidly screen and test for molecular players that are involved in the process of cell migration. The restriction enzyme-mediated intergration (REMI) technique is employed to tease out important molecules contributing to Dictyostelium cell adhesion and traction force during cell migration. The ease of performing targeted gene disruptions and mutagenesis in Dictystelium is useful in creating a clean background to study the interaction of proteins in multi-protein complexes such as the dynein-dynactin interaction, the role of dynein and myosin subunits, and the importance of phosphorylation in these molecular motors.
However, the limited structural details in Dictyostelium hinders further deciphering of the mechanistic aspects of how these motors work in generating a directed force to facilitate cell migration. To characterize these events, we have turned to the highly organized cytoskeleton of mammalian cells. Using GFP-tagged myosin, we examine the myosin-based contractile machinery and how the actomyosin system interact with microtubule network and the cell adhesion system. By duplicating the myosin regulatory light chain (RLC) phosphorylation site mutations initially characterized in Dictyostelium, we have probed the physiological significance of myosin phosphorylation during cell migration, cytokinesis, and isometric tension development using GFP-tagged mutant RLC. The advances made in the GFP technology has also made it possible for us to monitor signaling events governing myosin regulation in live cells. This is accomplished by the generation of a novel FRET-based biosensor, which allows us to simultaneously assess the localization, sequestration, and activity of myosin light chain kinase in vivo.
The results generated from genetic studies, biochemical characterization, and live cell imaging thus provide important foundation for our further understanding of the physiological roles of these molecular motors in whole animals. Our lab is currently also using molecular genetic techniques such as targeted gene disruption and in vitro mutagenesis in transgenic animals to manipulate myosin and dynein in vivo.