The Confocal Imaging Facility (CIF) of the NIA IRP has been in operation since October 2004. This facility started with one older model (Zeiss LSM 410) confocal system and has successfully added a Zeiss 710 confocal system in 2010, and the Zeiss LSM 880 with Airyscan in 2016. A state-of-the-art super-resolution system (Visitech VT-iSIM) was added in 2017. The expansion and upgrading of the CIF reflects the extensive use of confocal resources by intramural scientists at different levels of expertise, ranging from principal senior investigators through postdoctoral fellows and intramural research trainees. By FY17 over 150 different investigators have used the CIF, most of whom were trained by us. As a resource for all matters pertaining to microscopy and imaging, we have held scores of consultations with NIA Lab Chiefs, PIs and both intramural and extramural researchers to assist with their imaging and image analysis. We have helped users integrate imaging into their experimental systems, perform immunofluorescence studies, and troubleshoot problems encountered by investigators in their projects. The following imaging techniques have been used or were introduced at the CIF: (a) Precise sub-cellular localization and co-localization of proteins; (b) Investigation of intracellular and intra-nuclear protein trafficking; (c) Implementation of novel methodology to cause DNA damage to live cell DNA at the sub-micron level by continuous UV laser scanning; (d) Time lapse, FRAP and ratio-metric analysis of cellular processes in live cells; (e) Volumetric (3D) reconstruction of intracellular protein distribution using confocal or deconvolution techniques, and of organelles such as human pancreatic islets of Langerhans from optical Z-sections. (f) Devising novel, confocal-based methodology to investigate receptor activation using fluorescent ligands. (g) Devising novel, confocal-based and SIM-based methodologies to investigate mitochondrial distribution and function using immunofluorescence. (h) Integration of super-resolution microscope platforms such as the Airyscan and SIM as research tools for introduction into the NIA IRP. The importance of these techniques is emphasized in our recent research. We have followed the intracellular trafficking of several proteins, together with the precise sub-cellular localization and high-resolution co-localization of these proteins in multi-component protein complexes. Thus, in collaboration with the lab of Dr. Weeraratna (NIA and Wistar Institute), these methods have yielded very important characterizations of invasive melanoma cells: (a) defining Wnt5a (a protein that increases melanoma metastasis) interaction with syndecan which is via sugar chains, and was not amenable to immunonoprecipitation analysis, but which were proven to bind using immunofluorescence and confocal microscopy (See publications 2006-2011, also recently (O'Connell et al., Cancer Discov. 3(12):1378-93, 2013); (b) Using primarily immunofluorescence and confocal microscopy, we show that phosphorylation modifications of the tight junction protein claudin-1 cause its translocation to the cytoplasm and nucleus and that the sub-cellular localization of claudin-1 may dictate the metastatic capacity of melanoma cells. Our findings suggest that nuclear versus cytoplasmic expression of claudin-1 may become a valuable marker for diagnosis of malignant melanoma (French et al., Int. J. Med. Sci. 6:93-101, 2009); (c) Trafficking of EGFR-GFP showed endocytosis to late endosomes and lysosomes in cells expressing filamin A (an actin-binding protein), but not in cells that do not express filamin A, suggesting that filamin A contributes to activation and sorting of EGFR, an important member of the receptor tyrosine kinase family, that is implicated in oncogenesis (Fiori et al., Endocrinology 150:2551-60, 2009). Another important protein complex was discovered in a different system. In research done in collaboration with the lab of Dr. Biragyn (LI), we used in a T cell system a very nice three-color co-localization technique to provide quantitative evidence for the interaction of CD45 (a phosphatase that regulates Lck), Lck (a Src kinase) and GCR (glucocorticoid receptor) in a cell membrane protein complex that is important for the activation of T cells (Baatar et al., Brain Behav. Immunol. 23:1028-37, 2009). In intra-nuclear research we have assisted in defining the interactions of RNA binding proteins (Indig et al., PLoS One, 7:e35229, 2012) with our collaborators at the University of Maryland. CIF staff (Sarah Subaran, B.Sc.) contributed to a high-profile research paper published in the journal Nature Structural and Molecular Biology (Lee et al., 2010, Nat Struct Mol Biol 17:732-9). This research, performed as part of our ongoing collaboration with the lab of Dr. Gorospe (LCMB), involves imaging RNA binding proteins that form cytoplasmic structures called stress granules (Lee et al., 2012, PNAS 109:5750-5). These structures are defined exclusively by immunofluorescence, underlying the importance of imaging to the mission of the intramural research at the NIA. Subsequent CIF staff (Chris Moad, Christina Wohler and Logan George) have assisted in expanding this research into the nuclear-to-mitochondria trafficking of lncRNA RMRP, evident in several publications, for example, Noh et al., Genes & Dev 30:1224-39, 2016. Currently, the CIF is involved in several project with various labs at the NIA: (1) The investigation of Cannabinoid receptor binding in live cells (Drs. Bernier and de Cabo, TGB). Dr. Indig has devised a protocol to measure binding kinetics of GPR55 with live cell confocal microscopy. The investigation of Cannabinoid receptor binding in live cells has yielded a total of 4 peer-reviewed publications in collaboration with the CIF since 2013. (Krzysik-Walker et al., Mol. Pharmacol. 83(1):157-66, 2013; Paul et al., Biochem Pharmacol. 87(4):547-61, 2014; and Habicht et al., Anal Biochem. 484:154-61, 2015; Wnorowski et al., Cell Signal. 36:176-188, 2017). (2) The investigation of stress granules in mitochondria and nuclei using confocal and super- resolution techniques, together with the lab of Dr. Gorospe, LG. This study on RNA binding proteins and lncRNA has recently been published and validates the methodology developed for estimating mitochondrial density in human muscle (see below). This research has been reported in 2 recent publications (Osera et al., RNA Biol. 12(10) 1121-30, 2015 and Noh et al., Genes Dev. 30(10):1224-39, 2016). In another project with this lab, Dr. Indig has used Confocal microscopy to detect the expression of DPP4 on the cell surface of senescent cells and the elimination of senescent (DPP4+) cells from select cellular populations (Kim et al., Genes Dev. 31:1529-34, 2017). (3) Protein trafficking in neurons with Dr. Yao, LNS, demonstrates the importance of applying high resolution confocal and super-resolution techniques to the study of neurons and neural-derived exosomes. This research collaboration has yielded 2 publications since 2013. (Petralia et al., Neuromolec. Med. 15(1):49-60, 2013 and Eitan et al., Biol. Open, 5(8):1086-92, 2016). (4) Muscle mitochondria function and distribution, part of the CRB/BLSA Sarcopenia project (with Dr. Ferrucci, TGB, and Dr. Hari Shroff, NIBIB). This project is a major focal point at the CIF, which applies advanced imaging techniques including super-resolution microscopy to investigate mitochondrial distribution and function in young and aging human muscle tissue samples obtained by the GESTALT, BLSA and
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