7.61 Discussion #D3: Single Cell Assays: G Proteins and Ca++ DetectionAssigned Reading for Discussion #3, Wed.. October 13, 2004This set of readings addresses several different ways of analysing signalling pathwaysinindividual cells and single cell assays to monitor intracellular events. We have assigned threepapers ( marked by *) but we include for your information someother references addressingthe same issues.G PROTEINS*1. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G. and Wittig, B.(1991).Assignment of G-protein subtypes to specific receptors inducing inhibition ofcalcium currents. Nature 353:43-48.*2. Kleuss, C., Scherübl, H., Hescheler, J., Schultz, G. and Wittig, B. (1992). Different β-subunits determine G-protein interaction with transmembrane receptors. Nature358:424-426.These two papers describe and use methods for microinjection of antisense oligonucleotidesto ablate specific Gα or Gβ subunits and then test whether ion channel function alterations inresponse to specific hormones are affected.A third follow up paper extends this analysis to Gγ subunits.Kleuss, C., Scherübl, H., Hescheler, J., Schultz, G. and Wittig, B. (1993).Selectivity insignal transduction determined by _ subunits of heterotrimeric G proteins. Science259:832-834.Gollasch, M., Kleuss, C., Hescheler, J., Wittig, B . and Schultz, G. (1993). Gi2 andprotein kinase C are required for thyrotropin-releasing hormone-induced stimulation ofvoltage-dependent Ca2+ channels in rat pituitary GH3 cells. Proc. Natl. Acad. Sci.USA 90:6265-6269.This paper uses the same approach plus others to dissect a branched signaltransduction pathway involving several different Gα subunits, implicating different Gproteins in the different branches.++ DETECTION and GREEN FLUORESCENT PROTEIN*3. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. andTsien,RY. (1997). Fluorescent indicators for Ca2+ based on green fluorescent proteinsandcalmodulin. Nature 388: 882-887.This paper is one of a series of papers developing modified forms of GFP toassayvarious parameters at the single cell level.Rizzuto, R., Brini, M., DeGiorgi, F., Rosssi, R., Heim, R., Tsien, R.Y. and Pozzan, T.(1996). Double labelling of subcellular structures with organelle-targeted GFP mutantsinvivo. Curr. Biol. 6:183-188.An earlier paper in the sequence.7.61 Discussion 3 con’t. fall 04Other papers of relevance/interestScales, S.J., Pepperkok, R. and Kreis, T.E. (1997) Visualization of ER-to-Golgi Transport in living cellsreveals a sequential mode of action for COPI and COPII. Cell90: 1137- 111148.Use of similar methods to address the role of COPs I and II in intracellular transport.Kao, J.P.Y., Harootunian, A.T. and Tsien, R.Y. (1989). Photochemically generated cytosolic calciumpulses and their detection by Fluo-3*. J. Biol. Chem. 264:8179-8184.An even earlier development of one of the most widely used intracellular Ca++ sensorsJohn F. Presley, Carolyn Smith, Koty Hirschberg, Chad Miller, Nelson B. Cole, Kristien J. M. Zaal, andJennifer Lippincott-Schwartz Golgi Membrane Dynamics Mol. Biol. Cell 1998 9: 1617-1626.Photoactivatable GFP – This is likely to be used extensively in the future:Patterson GH, Lippincott-Schwartz J. A Photoactivatable GFP for Selective Photolabeling of Proteins andCells. Science. 2002 Sep
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