In the last year, 34 researchers have used the resources of the Laboratory of Cellular and Molecular Biology Microscopy Core. 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 Metabolism (Protein Section) and the Cell and Cancer Biology Branch. 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"""""""". Researchers working with Dr. Carol Clayberger have used the facility for the projects """"""""Regulation of RANTES Expression in T Lymphocytes"""""""" and """"""""Function of Granulysin"""""""". The Core has been involved with the project """"""""Cbl Proteins as Regulators of Tyrosine Kinase Signaling""""""""from Dr. Stanley Lipkowitz. Dr. Carole Parent's project, """"""""Signaling Events Regulating Chemotaxis"""""""" uses all of the Core instruments. Dr. Paul Randazzo has made extensive use of the Core for the projects """"""""Regulation of focal adhesions"""""""" and """"""""Turnover of invadopodia"""""""". The Core facility has assisted Dr.Jeffry Rubin with the project """"""""Secreted Frizzled-Related Proteins and Wnt Signaling"""""""". Dr. Ying Zhang uses Core instruments for the project """"""""Molecular Mechanisms of TGF-beta Signaling Pathway"""""""". In addition, the Core facility has been used by personnel working with Principal Investigators from other groups including work with Dr. Michael Bustin on the nucleosome binding protein HMGN1 and the role of heterochromatin formation in cell migration. This research usually involves the use of a Zeiss 510 Laser Scanning Confocal Microscope or a PerkinElmer UltraView Spinning Disk Confocal Microscope. Most of the users view immunofluorescent staining on fixed samples with the Zeiss 501 LSCM while the sjpinning disk confocal is used for live cell imaging. We have started to use our Total Internal Reflection Fluorescence (TIRF) microscope for PhotoActivation Localization Microscopy (PALM). This high resolution technique will allow us to determine the location of single proteins clustered in signaling complexes in T cells with an accuracy of around 20 nm, well below the diffraction limit of visible light. The TIRF system is also being used for the projects """"""""Regulation of focal adhesions"""""""" and """"""""Turnover of invadopodia"""""""" with Dr. Paul Randazzo.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICBC010978-04
Application #
8350120
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$336,966
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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. (2017) Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM. J Vis Exp :
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
Coussens, Nathan P; Hayashi, Ryo; Brown, Patrick H et al. (2013) Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells. Mol Cell Biol 33:4140-51
Sherman, Eilon; Barr, Valarie A; Samelson, Lawrence E (2013) Resolving multi-molecular protein interactions by photoactivated localization microscopy. Methods 59:261-9

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