Slides. Until recently, studies in this field of signal transduction have involved the "what" and "when" of signaling. Who talks to whom and for how long? With the advent of genetically encoded fluorescent proteins, it has become possible to monitor signaling events in living cells in real time. This has added the dimension of "where" to the study of cellular signaling. The slides and movies associated with this lecture, which is a part of "Cell Signaling Systems: A Course for Graduate Students," provide a survey of how green fluorescent protein (GFP)-tagged probes for signaling events have been used over the past seven years to elucidate new pathways, describe the kinetics of signaling events at the single cell level, and reveal upon which subcellular compartments these events take place. Some of the findings confirm previous ones using biochemical techniques and others have been surprising. Examples include those utilizing protein localization, relocalization, fluorescence recovery after photobleaching (FRAP), and fluorescence resonance energy transfer (FRET). The design of FRET probes is also considered. The detection of small GTPase signaling in living cells provides an example for exploring the creative and diverse ways investigators have developed to look at this system.

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Movie 1. Pleckstrin homology (PH) domain-green fluorescent protein (GFP) fusion proteins (PH-GFP) were used to monitor phosphatidylinositol-3,4,5-trisphosphate (PIP3) abundance in cellular membranes during chemotaxis of the slime mold Dictyostelium discoideum. This movie is Supplemental Video S4 entitled, "PH-GFP Localization in Chemotaxing Wild-Type Cells" from Iijima and Devreotes [Cell 109, 599-610 2002)]. This movie, which is mentioned in Slide 30, may also be downloaded from the Cell website (http://www.cell.com/cgi/content/full/109/5/599/DC1).

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Movie 2. PH-GFP was used to monitor changes in PIP3 abundance in D. discoideum lacking the lipid phosphatase PTEN. This movie is Supplemental Video S5 entitled, "PH-GFP Localization in Chemotaxing pten^— Cells" from Iijima and Devreotes [Cell 109, 599-610 2002)]. This movie, which is mentioned in Slide 30, may also be downloaded from the Cell website (http://www.cell.com/cgi/content/full/109/5/599/DC1).

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Educational Details

Learning Resource Type: PowerPoint slides and movies

Context: Undergraduate upper division, graduate, professional (degree program)

Intended Users: Teacher, learner

Intended Educational Use: Teach, learn, plan

Discipline: Biochemistry, Molecular Biology, Pharmacology, Structural Biology

Keywords: GTPases, Ras, GFP, FRET, FRAP

Technical Details Slides

Format: PowerPoint (ppt)

Size: 26.5 MB

Requirements: Microsoft PowerPoint

Technical Details Movies

Format: QuickTime Movie (mov)

Size: 3.1 MB, 3.5 MB

Requirements: QuickTime Player

Limits for Use

Cost: Free

Rights: This material may be downloaded for noncommercial, course-teaching purposes only, provided credit to STKE is included by listing the citation for the Teaching Resource.

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Citation: M.R. Philips, Imaging signal transduction in living cells with fluorescent proteins. Sci. STKE 2005, tr28 (2005).

© 2005 American Association for the Advancement of Science