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Green Fluorescent Protein in the Study of Neuronal Signaling Pathways

Leslie Blair¹, Kendra Bence‐Hanulec¹, John Marshall¹

¹Brown University, Providence, Rhode Island

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 5.16

DOI:

10.1002/0471142301.ns0516s14

Online Posting Date:

May, 2001

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Abstract

In recent years, techniques have been established for transiently co-transfecting cells with cDNA of the jellyfish green fluoresent protein (GFP), a reporter gene that encodes a non-toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co-transfecting with separate GFP and gene-of-interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene-of-interest and GFP cassettes. Also included is a method for the rapid, large-scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.

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Unit Introduction
Basic Protocol: Transfection of Green Fluorescent Protein and Gene-of-Interest Constructs
Support Protocol: Culturing Rat Cerebellar Granule Neurons
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Transfection of Green Fluorescent Protein and Gene-of-Interest Constructs

Materials

Rat pup cerebellar granule neurons (see Support Protocol)
Green fluorescent protein (GFP) cDNA
cDNA encoding gene of interest
0.25 M CaCl₂ (sterile), room temperature
2× BES-buffered saline (sterile; see recipe)
Growth medium (see recipe)
15-ml round-bottom polystyrene tubes (sterile)
Pasteur pipets (autoclaved)
Incubators, humidified 37°C, 3% and 5% CO₂

Support Protocol: Culturing Rat Cerebellar Granule Neurons

Materials

Silanizing solution (e.g., Sigmacote, Sigma)
0.1 to 1 mg/ml poly-d-lysine (mol. wt. >150,000 kDa) in sterile water
4- to 10-day-old rat pups
70% (v/v) ethanol
Ca²⁺, Mg²⁺-free HBSS/pen/strep, pH 7.4 (see recipe), ice-cold
0.5 g/liter trypsin solution (see recipe)
Fetal bovine serum (FBS; appendix 2A)
Growth medium (see recipe)
Tissue culture plates (35-, 60-, or 100-mm, depending on the final use) or glass coverslips in 35-mm plates
Dissecting tools (fine forceps, large forceps, fine dissecting scissors, iris scissors, large dissecting scissors), autoclaved
60-mm petri plates, sterile
#11 and #15 scalpels, sterile
15-ml polystyrene tubes, sterile
Incubator, humidified 37°C, 5% CO₂

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Figures

Figure 5.16.1

Example of a construct used to create a bicistronic transcript separately encoding GFP and the protein of interest. pTF-G6/IRES/GFP contains the coding sequences for SuperGlo sg25-GFP (Qbiogene) and the GluR6 kainate receptor (gift of Drs. J. Boulter and S. Heinemann). The cytomegalovirus (CMV) promoter-enhancer was used to ensure a high level of expression in mammalian cells. To drive individual translation of GFP and receptor proteins from the single bicistronic mRNA, the construct also contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus, located between the GluR6 and GFP coding sequences. Constructed by T. Fukushima.

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Figure 5.16.2

Transfection of primary neurons and applications to studying signaling pathways. (A) Cultured cerebellar granule neurons were transiently co-transfected with cDNAs encoding a p85 subunit of phosphatidylinositol-3¢-kinase (PI3K) and GFP. Overlay of a fluorescence micrograph superimposed on the Nomarski image of the field. Scale bar, 10 µm. (B) Expression of an inactive p85 subunit blocks insulin-like growth factor 1 (IGF-1) potentiation of neuronal L-type calcium channels (upper trace, currents recorded before and 10 and 60 sec after addition of IGF-1 superimposed), but overexpression of wild-type (wt) p85 results in dramatically increased rapid IGF-1 responses (lower trace). Arrowheads indicate current after IGF-1 addition. Reprinted from Blair and Marshall (1997) with permission from Neuron. (C) Neuronal survival via IGF-1 modulation of L-type calcium channels is mediated by the serine/threonine kinase, Akt. Left panels, wt Akt transfectants as indicated by GFP fluorescence. Right panels, the same fields showing propidium iodide (PI) labeling of all neurons to reveal DNA; solid arrowheads indicate transfectants with the condensed chromatin characteristic of apoptotic cells and arrows indicate healthy transfectants; open arrowheads indicate examples of untransfected apoptotic cells. In the upper panels, wt-Akt-transfected neurons survive much better than their untransfected neighbors. In the lower panels, in medium containing an L-type calcium channel inhibitor, survival of transfected and untransfected neurons is similar. Reprinted from Blair et al. (1999a) with permission from J. Neuroscience.

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Figure 5.16.3

Transfection of granule neurons with SAP97, a potential scaffolding protein for co-anchoring ion channels and signaling intermediates, directly tagged with GFP. Direct tagging allows subcellular localization of the SAP97 protein. Punctate appearance of the GFP tag along the neurites indicates restriction of SAP97 to discrete assemblages; the GFP tag also indicates cytoplasmic SAP97 expression in the soma. Micrograph by Dr. V. Kumaresan.

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Literature Cited

Literature Cited
	Arbert-Engels, C., Tartare-Deckert, S., and Eckhart, W. 1999. C-terminal Src kinase associates with ligand-stimulated insulin-like growth factor-1 receptor. J. Biol. Chem. 274:5422-5428.
	Blair, L.A.C. and Marshall, J. 1997. IGF-1 modulates N and L calcium channels in a PI 3-kinase-dependent manner. Neuron 19:421-429.
	Blair, L.A.C., Bence-Hanulec, , K.K., Mehta, S., Franke, T., Kaplan, D., and Marshall, J. 1999a. Akt-dependent potentiation of L channels by IGF-1 is required for neuronal survival. J. Neurosci. 19:1940-1951.
	Blair, L.A.C., Bence-Hanulec, K.K., and Marshall, J. 1999b. The jellyfish green fluorescent protein: A tool for studying ion channels and second messenger signalling in neurons. Methods Enzymol. 302:213-225.
	Chen, C. and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752.
	Cohick, W.S. and Clemmons, D.R. 1993. The insulin-like growth factors. Annu. Rev. Physiol. 55:131-153.
	Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20:448-455.
	Delbono, O., Renganathan, M., and Messi, M.L. 1997. Regulation of mouse skeletal muscle L-type Ca²⁺ channel by activation of the insulin-like growth factor-1 receptor. J. Neurosci. 17:6918-6928.
	Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R., and Greenberg, M.E. 1997. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275:661-665.
	Finkbeiner, S. and Greenberg, M.E. 1996. Ca⁺²-dependent routes to Ras: Mechanisms for neuronal survival, differentiation, and plasticity Neuron 16:233-236.
	Franke, T.F., Yang, S.-I., Chan, T.O., Datta, K., Kazlauskas, A., Morrison, D.K., Kaplan, D.R., and Tsichlis, P.N. 1995. The protein kinase encoded by the Akt proto-oncogene is a target of PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727-736.
	Goslin, K. and Banker, G. 1991. Rat hippocampal neurons in low density. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.) pp. 251-282. MIT Press, Cambridge, Mass.
	Heim, R. and Tsien, R. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6:178-182.
	Hilborn, M.D., Vaillancourt, R.R., and Rane, S.G. 1998. Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway. J. Neurosci. 18:590-600.
	Kim, B., Cheng, H.L., Margolis, B., and Feldman, E.L. 1998. Insulin receptor substrate 2 and Shc play different roles in insulin-like growth factor I signaling. J. Biol. Chem. 273:34543-34550.
	Klint, P., Kanda, S., Koog, Y., and Claesson-Welsh, L. 1999. Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation. Oncogene 18:3354-3364.
	Marshall, J., Molloy, R., Moss, G.W.J., Howe, J.R., and Hughes, T.E. 1995. The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function. Neuron 14:211-215.
	Messer, A. 1972. The maintenance and identification of mouse cerebellar granule cells in monolayer culture. Brain Res. 130:1-12.
	Palm, G.J., Zdanov, A., Gaitanaris, G.A., Stauber, R., Pavlakis, G.N., and Wlodawer, A. 1997. The structural basis for spectral variations in green fluorescent protein. Nature Struct. Biol. 4:361-365.
	Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229-233.
	Qu, C.K., Yu, W.M., Azzarelli, B., and Feng, G.S. 1999. Genetic evidence that Shp-2 tyrosine phosphatase is a signal enhancer of the epidermal growth factor receptor in mammals. Proc. Natl. Acad. Sci. U.S.A. 96:8528-8533.
	Stauber, R.H., Horie, K., Carney, P., Hudson, E.A., Tarasova, N.I., Gaitanaris, G.A., and Pavlakis, G.N. 1998. Development and applications of enhanced green fluorescent protein mutants. BioTechniques 24:462-472.
	Wachter, R.M., King, B.A., Heim, R., Kallio, K., Tsien, R.Y., Boxer, S.G., and Remington, S.J. 1997. Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. Biochemistry 36:9759-9765.
	Wang, J., Dai, H., Yousaf, N., Moussaif, M., Deng, Y., Boufelliga, A., Swamy, O.R., Leone, M.E., and Riedel, H. 1999. Grb-10, a positive, stimulatory signaling adapter in platelet-derived growth factor BB-, insulin-like growth factor I-, and insulin-mediated mitogenesis. Mol. Cell. Biol. 19:6217-6228.
	Wu, H., Reuver, S.M., Kuhlendahl, S., Chung, W.J., and Garner, C.C. 1998. Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane. J. Cell Sci. 111:2365-2376.