Last year, 31 researchers used the resources of the Laboratory of Cellular and Molecular Biology Microscopy Core. Almost all of the Principal Investigators in the Laboratory of Cellular and Molecular Biology have projects that involve the Core facility. Dr. Lawrence Samelson uses Core resources for the project Biochemical basis of T cell activation. Dr. Paul Randazzo has made extensive use of the Core for the projects Regulation of focal adhesions and Turnover of invadopodia. Dr. Stavroula Mili has used Core instruments for the project Regulation and functions of localized RNAs. Dr. Ying Zhang uses Core instruments for the project Molecular mechanisms of TGF-beta signaling pathways. Dr. Ramiro Iglesias-Bartolome uses Core instruments for two projects, Signaling pathways regulating stem cell fate decisions and G-protein-coupled receptor signaling in cancer development and treatment. While most of the researchers come from the Laboratory of Cellular and Molecular Biology, the Core has been used by scientists from the Laboratory of Immune Cell Biology of the National Cancer Institute, the Cancer and Inflammation Program of the National Cancer Institute, the Women's Malignancies Branch of the National Cancer Institute, the Retinal Cell Biology and Degeneration Section of the National Eye Institute, the Genetic Disease Research Branch of the National Human Genome Research institute and The University of Maryland Department of Physics. In addition, the Core facility has performed collaborative work with outside investigators such as Robert Rottapel of the Ontario Institute for Cancer Research at the University of Toronto. Most of the users view immunofluorescent staining on fixed samples with a Leica laser scanning confocal microscope. A spinning disk confocal is generally used for live cell imaging and all of our systems are equipped with on-stage incubators so that live cell imaging can be done on any microscope. We routinely use our TIRF microscope for direct Stochastic Optical Reconstruction Microscopy (dSTORM). This Single Molecule Localization technique allows us to determine the position of single proteins with a localization error of around 10 nm using fiducial markers developed by the Biochemistry & Biophysics Center at National Heart Lung and Blood Institute. We are currently working on developing statistical methods to analyze the positions of 6 different proteins involved in T cell activation. The Laboratory of Cellular and Molecular Biology Microscopy Core contains resources for analyzing and quantifying image data. We recently obtained a new state-of-the-art image processing station for large 4D data sets, including analysis of single molecule data. Core personnel also provide information on new imaging technologies to NCI researchers. This year the LCMB Core personnel presented a report on optogenetics to the LCMB researchers so that they could add these techniques to their current research projects using Core instruments.
| Guittard, Geoffrey; Dios-Esponera, Ana; Palmer, Douglas C et al. (2018) The Cish SH2 domain is essential for PLC-?1 regulation in TCR stimulated CD8+ T cells. Sci Rep 8:5336 |
| Yi, Jason; Manna, Asit; Barr, Valarie A et al. (2017) Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM. J Vis Exp : |
| Dutta, Debjani; Barr, Valarie A; Akpan, Itoro et al. (2017) Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol 18:196-204 |
| Barr, Valarie A; Yi, Jason; Samelson, Lawrence E (2017) Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy. Methods Mol Biol 1584:183-206 |
| Yi, Jason; Manna, Asit; Barr, Valarie A et al. (2016) madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy. Mol Biol Cell 27:3591-3600 |
| Sherman, Eilon; Barr, Valarie A; Merrill, Robert K et al. (2016) Hierarchical nanostructure and synergy of multimolecular signalling complexes. Nat Commun 7:12161 |
| Barr, Valarie A; Sherman, Eilon; Yi, Jason et al. (2016) Development of nanoscale structure in LAT-based signaling complexes. J Cell Sci 129:4548-4562 |
| Balagopalan, Lakshmi; Kortum, Robert L; Coussens, Nathan P et al. (2015) The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters. J Biol Chem 290:26422-9 |
| Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal et al. (2014) In vivo functional mapping of the conserved protein domains within murine Themis1. Immunol Cell Biol 92:721-8 |
| Sherman, Eilon; Barr, Valarie; Samelson, Lawrence E (2013) Super-resolution characterization of TCR-dependent signaling clusters. Immunol Rev 251:21-35 |
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