Encoding Green Fluorescent Protein Research Articles

Evidence for the formation of functionally distinct alphabetagammaepsilon GABA(A) receptors.

1. We transiently introduced the human GABA(A) receptor epsilon subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing alpha1beta3gamma2 receptors (WSS-1 cells) to establish whether the subunit competes with the gamma2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the epsilon subunit cDNA. 2. The epsilon subunit did not change the potency of GABA: the GABA EC(50) was 34 +/- 6 microM in control WSS-1 cells and 37 +/- 6 microM in cells expressing the epsilon subunit. The introduction of the epsilon subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 +/- 0.2 nA in control cells to 0.9 +/- 0.2 nA in cells expressing the epsilon subunit (P < 0.05). 3. The epsilon subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from epsilon subunit-containing WSS-1 cells exhibited spontaneously opening GABA(A) channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the epsilon subunit did not alter the GABA-evoked single-channel cord conductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 microM) and the benzodiazepine flunitrazepam (1 microM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The epsilon subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the epsilon subunit had no effect on the ability of propofol (3-30 microM) relative to GABA (1 mM) to activate GABA(A) receptors in WSS-1 cells. High concentrations of propofol (> or = 100 microM) produced a more marked desensitization of GABA(A) receptor activity in WSS-1 cells transfected with cDNA for the epsilon subunit than in control cells. 6. There was no difference in the potency of Zn(2+) as an inhibitor of currents recorded from control cells (IC(50) = 165 +/- 34 microM) or cells expressing the epsilon subunit (IC(50) = 179 +/- 11 microM). 7. GABA-activated currents recorded both from control cells and cells expressing the epsilon subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification. 8. The introduction of the epsilon subunit changes the functional properties of GABA(A) receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of alpha, beta, gamma and epsilon subunits.

Structure and Targeting of RyR1: Implications from Fusion of Green Fluorescent Protein at the Amino-Terminal

In skeletal muscle, an anterograde signal from the dihydropyridine receptor (DHPR) to the ryanodine receptor (RyR1) is required for excitation–contraction (EC) coupling and a retrograde signal from RyR1 to the DHPR regulates the magnitude of the calcium current carried by the DHPR. As a tool for studying biosynthesis and targeting, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the amino terminal of RyR1 and expressed it in dyspedic myotubes. The GFP-RyR1 was present in a restricted domain near the nucleus injected with cDNA and was fully functional, which places constraints on the location of the amino terminal in the folded structure of RyR1.

A Carboxyl-terminal Region Important for the Expression and Targeting of the Skeletal Muscle Dihydropyridine Receptor

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

Additional Transduction Events after Subretinal Readministration of Recombinant Adeno-Associated Virus

Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.

Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina.

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV. CMV.EGFP. The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.

A Functional DNA Binding Domain Is Required for Growth Hormone-induced Nuclear Accumulation of Stat5B

The mechanisms regulating the cellular distribution of STAT family transcription factors remain poorly understood. To identify regions of Stat5B required for ligand-induced nuclear accumulation, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the N terminus of Stat5B and performed site-directed mutagenesis. When co-expressed with growth hormone (GH) receptor in COS-7 cells, GFP-Stat5B is tyrosyl-phosphorylated, forms dimers, and binds DNA in response to GH in a manner indistinguishable from untagged Stat5B. In multiple cell types, laser scanning confocal imaging of GFP-Stat5B co-expressed with GH receptor shows that GFP-Stat5B undergoes a rapid, dramatic accumulation in the nucleus upon GH stimulation. We introduced alanine substitutions in several regions of Stat5B and assayed for GH-dependent nuclear localization. Only the mutation that prevented binding to DNA (466VVVI469) abrogated GH-stimulated nuclear localization. This mutant fusion protein is tyrosyl-phosphorylated and dimerizes in response to GH. These results suggest that either high affinity binding to DNA contributes to nuclear accumulation of Stat5B or that this region is crucial for two functions, namely accumulation of Stat5B in the nucleus and DNA binding. Thus, we have identified a mutant Stat5 defective in nuclear localization despite its ability to be tyrosyl-phosphorylated and to dimerize.

Developmental Transitions and Dynamics of the Cortical ER of Arabidopsis Cells Seen with Green Fluorescent Protein

Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.

Mice with a Fluorescent Marker for Interleukin 2 Gene Activation

Production of interleukin (IL)-2 by T lymphocytes is one of the earliest events during immune response. A mutant mouse strain was generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein (GFP). In this model, GFP fluorescence is readily detectable upon T cell activation and is mostly coexpressed with IL-2 at the single cell level. Thus, individual activated T cells can express the IL-2 gene biallelically. Upon stimulation through the T cell antigen receptor, CD4 + cells separate into distinct GFP + and GFP − populations, both of which are capable of differentiating into either Th1 or Th2 effectors. These mice allow noninvasive detection of IL-2 production by single cells and analysis of the subsequent differentiative fate of these cells as an immune response develops.