The Confocal Imaging Facility (CIF) of the NIA IRP has been in operation since October 2004. This facility has one older model (Zeiss LSM 410) confocal system with low usage by IRP investigators. The NIA CIF has been managed and overseen with the expansion and successfully integration of a state of the art Zeiss LSM 510 Meta confocal system, updating the Zeiss Axioskop fluorescent microscope MCID/QImage analysis system and using Metamorph/Coolsnap imaging and analysis system. This has resulted in extensive collaboration with intramural scientists at different levels of expertise ranging from principal senior investigators through postdoctoral fellows and intramural research trainees. We have trained over thirty investigators to be independent users of the confocal microscope and have held scores of consultations with NIA researchers to assist with their imaging and image analysis. We have helped users integrate imaging into their experimental systems and in troubleshooting problems. This has resulted in an increase in the use of immunofluorescence and imaging at the IRP, as attested by the widespread use of the CIF last year by over 60 researchers, ranging from post-bacs to senior investigators. The following techniques have been used or introduced at the CIF: (a) Use of confocal microscopy for precise sub-cellular localization and co-localization of proteins;(b) Advanced immunological, biochemical and imaging techniques required for the investigation of intracellular and intranuclear protein trafficking;(c) Development of methodology to cause DNA damage to live cell DNA at the sub-micron level by use of the continuous scanning UV laser of the LSM 510 Meta system;(d)Time-lapse, FRAP and ratio-metric analysis of cellular processes in live cells using the 510 Meta confocal microscope;(e) Volumetric (3D) reconstruction of intracellular protein distribution using confocal or deconvolution techniques. The importance of these techniques is emphasized in the recent research of the CIF and our collaborators (see Bibliography below, 1-6). 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, 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 (6);(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 (3);(c) Trafficking of EGFR-GFP showed endocytosis to late endosomes and lysosomes in cells expressing flamin A (an actin-binding protein), but not in cells that do not express flamin A, suggesting that flamin A contributes to activation and sorting of EGFR, an important member of the receptor tyrosine kinase family, that is implicated in oncogenesis(2). Another important protein complex was discovered in a different system. In a T cell system we used a very nice three-color co-localization technique to providing 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 (4).
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