The long term goal of this grant is to advance understanding of chemotaxis, the ability of cells to sense chemical gradients and move directionally. Studies oi Dictyostelium discoideum, with its accessible biochemistry, cell biology, and genetics, have contributed enormously to the current understanding of chemotaxis. Briefly, in Dictyostelium and human neutrophils, chemotaxis is mediated by GPCRs linked to specific heterotrimeric G-proteins, which trigger downstream signaling events to occur selectively on the side of the cell facing the higher concentration of chemoattractant. For example, the accumulation of phosphatidylinositol 3,4, 5-tris phosphate (PIP3) is sharply localized to the leading edge of the cell. Recent studies have shown that PIP3 acts in parallel with other signaling events in a network that biases the motility machinery of the cell. In the Progress Report, we describe our studies of tsunami which plays a key role in cell polarity. We define me central roles of pianissimo (piaA), a subunit of Tor Complex 2 (TorC2), and a phospholipase A2, plaA. We report single molecule imaging studies of receptors and G-proteins revealing the earliest steps in gradient sensing. We describe the results of a large forward genetic screen that uncovered over twenty novel chemotaxis genes. We also report on a genomic library complementation approach to identify chemically-induced mutants. The latter two projects, involving considerable risk and long term commitment, were made possible by the MERIT award. The plans for the second half of the grant are presented. We will continue to unravel the network of signaling pathways that mediate chemotaxis, exploiting the ability to combine gene disruptions in Dictyostelium, along with careful biochemical and cell biological experiments. We will ask whether key elements of the pathways are conserved in neutrophils. To define the roles of the new chemotaxis genes we have identified, we will assess a series of well-characterized physiological responses associated with chemotaxis such as PIP3 production, actin polymerization, and phosphorylation of PKB substrates and carry out and analyze time-lapse images of the cells undergoing directed migration. We will pursue the single molecule imaging studies to achieve and refine direct imaging individual G-protein activation events, hopefully creating as powerful an experimental tool as single channel recording of ion channel openings.

Public Health Relevance

Chemotaxis mediates many physiological processes including embryogenesis, immune response, wound healing, and stem cell homing and plays a key role in the pathology of inflammatory diseases and cancer metastasis. A detailed understanding of chemotaxis, which involves links between signal transduction and cell motility networks, would provide an incredibly powerful tool for designing rational interventions for improving health and defeating disease.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM028007-35
Application #
8676809
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Gaillard, Shawn R
Project Start
Project End
Budget Start
Budget End
Support Year
35
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Tang, Ming; Wang, Mingjie; Shi, Changji et al. (2014) Evolutionarily conserved coupling of adaptive and excitable networks mediates eukaryotic chemotaxis. Nat Commun 5:5175
Nguyen, H N; Afkari, Y; Senoo, H et al. (2014) Mechanism of human PTEN localization revealed by heterologous expression in Dictyostelium. Oncogene 33:5688-96
Cai, Huaqing; Katoh-Kurasawa, Mariko; Muramoto, Tetsuya et al. (2014) Nucleocytoplasmic shuttling of a GATA transcription factor functions as a development timer. Science 343:1249531
Artemenko, Yulia; Lampert, Thomas J; Devreotes, Peter N (2014) Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes. Cell Mol Life Sci 71:3711-47
Nguyen, Hoai-Nghia; Yang, Jr-Ming; Afkari, Yashar et al. (2014) Engineering ePTEN, an enhanced PTEN with increased tumor suppressor activities. Proc Natl Acad Sci U S A 111:E2684-93
Huang, Chuan-Hsiang; Tang, Ming; Shi, Changji et al. (2013) An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration. Nat Cell Biol 15:1307-16
Shi, Changji; Huang, Chuan-Hsiang; Devreotes, Peter N et al. (2013) Interaction of motility, directional sensing, and polarity modules recreates the behaviors of chemotaxing cells. PLoS Comput Biol 9:e1003122
Tang, Ming; Iijima, Miho; Kamimura, Yoichiro et al. (2011) Disruption of PKB signaling restores polarity to cells lacking tumor suppressor PTEN. Mol Biol Cell 22:437-47
Cai, Huaqing; Devreotes, Peter N (2011) Moving in the right direction: how eukaryotic cells migrate along chemical gradients. Semin Cell Dev Biol 22:834-41
Artemenko, Yulia; Swaney, Kristen F; Devreotes, Peter N (2011) Assessment of development and chemotaxis in Dictyostelium discoideum mutants. Methods Mol Biol 769:287-309

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