Molecular and Cell Biology / Cell and Developmental Biology
Education: Ph.D. University of Wisconsin
Research Interests: The actin cytoskeleton provides the structural framework for cells and dynamic changes in actin filaments are required for many cell processes including motility, cytokinesis, phagocytosis, and endocytosis. Our lab is studying the signal transduction pathways that lead to changes in the actin cytoskeleton and the actin binding proteins that organize the network of filaments. Four major projects are ongoing:
1. Actin binding proteins in Cell Dynamics and Disease: Once actin filaments are polymerized, actin binding proteins organize the filaments into functional arrays. We are studying several actin binding proteins that create orthogonally cross-linked actin networks to determine the contribution of each to cytoskeletal structure. Fimbrin, filamin and alpha-actinin are being fused to GFP and expressed in cells and live cell time-lapse confocal microscopy is used to determine how their localization is controlled in Dictyostelium amoebae and mouse macrophages. Mutant versions of these proteins that are known to cause human disease are being expressed in cell lines to create in vitro models of these diseases.
2. Phagocytosis and Apoptosis lead to Silicosis: The process of phagocytosis is important both for immune defense against invading organisms and for clearance of apoptotic cells. However, when cells phagocytose silica particles, apoptosis is initiated and the cells die. When silica dust is inhaled, it can cause an inflammation of the lungs called silicosis in humans. We are studying the mechanism by which cells take up silica and the signaling events that lead to apoptosis.
3. Myosin motor function: The role of the motor protein myosin II in non-muscle cell physiology is still unclear. We have created a light chain and heavy chain myosin mutants to examine their localization and function in chemotaxis and development. The results have shed new light on the distinction between myosin’s role as an actin cross-linking protein and its role as a motor protein. We are currently collaborating with Dr. Juliet Lee’s laboratory to measure the forces wild-type and mutant cells apply to surfaces during motility.
4. The Genetics of Macropinocytosis: The process of macropinocytosis is the uptake of extracellular fluid into very large vesicles through an actin-mediated process. This process is used by immune cells to process antigens and by HIV as a means to infect cells. Mutants defective in macropinocytosis have been isolated and are presently being characterized.
Each project combines molecular genetic approaches with digital imaging and confocal microscopy to study the behavior of normal and mutant cells. GFP-fusion constructs that allow visualization of numerous signaling and structural proteins allow us to follow dynamic processes in living cells.
Xu, X., Lee, E., Chen, T., Kuczmarski, E., Chisholm. R. L. and Knecht, D.A.. (2001) During multicellular migration, Myosin II serves a structural role independent of its motor function. Develop. Biol., 232:255-264.
Rupper A, Lee K, Knecht D, Cardelli J. (2001) Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium. Mol Biol. Cell. 12:2813-2824.
Laevsky, G. and Knecht, D. A. (2001) Under-Agarose Folate Chemotaxis of Dictyostelium Amoebae in Permissive and Mechanically Inhibited Conditions. Biotechniques, 31:1140- 1149.
Hadjout, N., Laevsky, G., Knecht, D. A. and Lynes, M.A. (2001) Automated Real-time Measurement of Chemotactic Cell Motility. Biotechniques, 31:1130-1138.
Pang, K.M., Dingermann, T. and Knecht, D. A. (2002) Regulated Expression of Myosin II Heavy Chain and RacB Using an Inducible tRNA Suppressor Gene. Gene, 277:187-197.
Maselli A, Laevsky G, Knecht DA. (2002) Kinetics of binding, uptake and degradation of live fluorescent (DsRed) bacteria by Dictyostelium discoideum. Microbiology,148:413-420.
Lee, E. and Knecht, D.A. (2002) Visualization of Actin Dynamics During Macropinocytosis and Exocytosis. Traffic, 3:186-192.
Olazabal I, Caron E, May R, Schilling K, Knecht D, Machesky L. (2002) Rho-Kinase and Myosin-II Control Phagocytic Cup Formation during CR, but Not FcgammaR, Phagocytosis. Curr Biol. 12: 1413-1418.
Lee, E., Seastone, D. J., Harris E., Cardelli, J. A., and Knecht D. A. (2003) RacB regulates cytoskeletal function in Dictyostelium. Eukayrotic Cell, 2:474-485.
Laevsky G, Knecht DA. (2003) Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments. J Cell Sci. 116:3761-3770.
Thibodeau, M.S., Giardina, C., Knecht, D.A., Helble, J., and. Hubbard A.K. (2004) Silica-induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity. Tox. Sci, 80: 34-48.
Scott, C.C., Dobson, W., Botelho, R.J., Coady-Osberg, N., Chavrier, P., Knecht, D.A., Heath, C., Stahl, P. and Grinstein, S.,(2005) Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J. Cell Biol., 169:139-149.
Yin, X., Knecht, D.A., Lynes, M.A (2005) Metallothionein mediates leukocyte chemotaxis BMC Immunology, 6:21-33.
Hadjout,N., Yin, Y., Knecht, D.A. and Lynes, M.A. (2007) Automated real-time measurements of leukocyte chemotaxis, J Immunol Methods. 2007 320:70-80.
Lombardi ML, Knecht DA, Dembo M, Lee J. (2007) Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement. J Cell Sci. 120:1624-1634.
Lombardi, M. L., Knecht, D. A., and Lee J. (2008) Mechano-chemical Signaling Maintains the Rapid Movement of Dictyostelium Cells. Exp. Cell Res., 314:1850-1859.
Washington, R. and Knecht, D.A. (2008) Actin-binding domains direct actin-binding proteins to different cytoskeletal locations. BMC Cell Biology, 9:1-16.
Gilberti ,R., Joshi, G., and Knecht, D.A. (2008) The Phagocytosis of Crystalline Silica Particles by Macrophages, American Journal of Respiratory Cell and Molecular Biology PMID: 18556590 39(5):619-627.
Tang L, Franca-Koh J, Xiong Y, Chen MY, Long Y, Bickford RM, Knecht DA, Iglesias PA, Devreotes PN. (2008) tsunami, the Dictyostelium homolog of the Fused kinase, is required for polarization and chemotaxis. Genes Dev. 22:2278-2290.
Veltman DM, Lemieux MG, Knecht DA, Insall RH (2014) PIP(3)-dependent macropinocytosis is incompatible with chemotaxis. J Cell Biol 204: 497-505.
Lemieux, M. G., Janzen, D., Hwang, R., Roldan, J., Jarchum, I. and Knecht, D. A. (2014) Visualization of the actin cytoskeleton: different F-actin-binding probes tell different stories. Cytoskeleton (Hoboken) 71, 157-16930. Joshi GN,
Knecht DA (2013) Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages. Apoptosis 18: 271-285.
Andrew J. Muinonen-Martin, Olivia Susanto, Qifeng Zhang, Elizabeth Smethurst, William J. Faller, Douwe M. Veltman, Gabriela Kalna, Colin Lindsay, Dorothy C. Bennett, Owen J. Sansom, Robert Herd, Robert Jones, Laura M. Machesky, Michael J. O. Wakelam, David A. Knecht, Robert H. Insall (2014) Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal. PLoS Biology. DOI: 10.1371/journal.pbio.1001966
Veltman, D. M., Lemieux, M. G., Knecht, D. A. and Insall, R. H. (2014) PIP(3)-dependent macropinocytosis is incompatible with chemotaxis. J Cell Biol 204, 497-505
Gilberti, R. M. and Knecht, D. A. (2015) Macrophages phagocytose nonopsonized silica particles using a unique microtubule-dependent pathway. Mol Biol Cell 26, 518-529
Joshi, G. N., Goetjen, A. M. and Knecht, D. A. (2015) Silica particles cause NADPH oxidase-independent ROS generation and transient phagolysosomal leakage. Mol Biol Cell 26, 3150-3164
Tweedy, L., Knecht, D. A., Mackay, G. M. and Insall, R. H. (2016) Self-Generated Chemoattractant Gradients: Attractant Depletion Extends the Range and Robustness of Chemotaxis. PLoS Biol 14, e1002404
Joshi, G. N., Gilberti, R. M. and Knecht, D. A. (2017) Single Cell Analysis of Phagocytosis, Phagosome Maturation, Phagolysosomal Leakage, and Cell Death Following Exposure of Macrophages to Silica Particles. Methods Mol Biol 1519, 55-77
Paschke, P., Knecht, D. A., Silale, A., Traynor, D., Williams, T. D., Thomason, P. A., Insall, R. H., Chubb, J. R., Kay, R. R. and Veltman, D. M. (2018) Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum. PLoS One 13, e0196809
|Mailing Address||91 North Eagleville Road, Unit 3125, Storrs, CT 06269-3125|
|Office Location||Biology/Physics Building 307|