1Department of Medicine and 2Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637
Submitted 19 March 2004 ; accepted in final form 21 May 2004
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ABSTRACT |
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intracellular calcium; cameleon; fluorescence resonance energy transfer
Expression of these probes in intact mammalian tissues has proven difficult. One of the major impediments has been the lack of an effective method to deliver biosensor cDNA to cells within a living mammal; this cannot be accomplished effectively and reproducibly with liposomal, electroporation, viral, or ballistic methods but conceivably could be attained using a transgenic approach. Biosynthetic fluorescent cameleon and camgaroo Ca2+ sensors based on green fluorescent protein (GFP) and the Ca2+ binding protein, calmodulin (13, 17), have been engineered to label specific populations of cells within intact tissues in nonmammalian transgenic organisms (Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio) (5, 911, 16, 20, 21). No mammalian transgenic expression of a FRET-based Ca2+ biosensor, however, has been reported. In this paper we demonstrate transgenic expression of yellow cameleon3.3er (YC3.3er), a biosynthetic indicator of endoplasmic reticulum Ca2+ (6), in endocrine cells of mouse pancreas. Our studies demonstrate the feasibility of applying FRET biosensor imaging technology to transgenic mammalian cell systems and suggest that this approach may be used for real-time visualization of cell signaling in living tissues.
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MATERIALS AND METHODS |
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MIP-YC3.3-er transgenic mice. The purified transgene DNA was microinjected into the pronuclei of CD-1 mice at the Transgenic Mouse/ES Core Facility of the University of Chicago Diabetes Research and Training Center. The transgene was maintained on the CD-1 background, and mice were housed under specific pathogen-free (SPF) conditions with free access to food and water. Tail DNA from potential founder mice was screened for the presence of the transgene by PCR using forward and reverse primers 5'-GACAACCACTACCTGAGCTAC-3' and 5'-ACTGGGCTTACATGGCGATACTC-3', respectively. Whole pancreata of F1 progeny were visualized with an Olympus SZX12 stereomicroscope (Olympus, Melville, NY) in bright-field and fluorescence illumination modes. All the procedures involving mice were approved by the University of Chicago Institutional Animal Care and Use Committee.
Glucose tolerance testing. Intraperitoneal glucose tolerance tests were performed after a 4-h fast. Blood was sampled from the tail vein before and 30, 60, 90, and 120 min after intraperitoneal injection of 2 mg/g body wt of dextrose. Glucose levels were measured using a Precision Q.I.D. Glucometer (MediSense, Waltham, MA).
Isolation of islets of Langerhans. Pancreatic islets were isolated as described (7). Briefly, the pancreas was inflated with a solution containing 0.3 mg/ml collagenase (Type XI; Sigma, St. Louis, MO) in Hanks' balanced salt solution, injected via the pancreatic duct. The inflated pancreas was removed, incubated at 37°C for 10 min, and shaken vigorously to disrupt the tissue. After differential centrifugation through a Ficoll gradient to separate islets from acinar tissue, the islets were washed and placed on 25-mm glass coverslips. Islets were cultured in RPMI 1640 supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated in a humidified incubator at 37°C in 95% air and 5% CO2. All imaging studies were performed 25 days after isolation.
Immunofluorescence microscopy.
Sections (6 µm in thickness) from paraffin-embedded pancreatic tissue were incubated with monoclonal anti-GFP antibody (Sigma) to detect the expression of MIP-YC3.3-er, polyclonal anti-porcine insulin antibody to identify -cells, and a combination of polyclonal anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide (PP) antibodies to identify
-,
-, and PP cells, respectively (DakoCytomation, Carpinteria, CA). The endoplasmic reticulum (ER) was labeled with a polyclonal anti-glucose-regulated protein 94 (GRP94) antibody (Stressgen, Victoria, BC, Canada). Biotin-streptavidin-conjugated anti-mouse IgG and Cy2-conjugated streptavidin anti-mouse and Texas Red-conjugated anti-guinea pig/rabbit IgG secondary antibodies were used for MIP-YC3.3-er and insulin, respectively. Cy5-conjugated secondary antibody was used for non-
-cells and GRP94 (Jackson ImmunoResearch Laboratory, West Grove, PA). The stained sections were visualized with a Leica SP2 AOBS confocal microscope.
Confocal microscopy of isolated islets. Laser scanning confocal images were collected on a Leica SP2 AOBS spectral confocal microscope system using a x63 NA1.3 glycerol objective, viewing coverslip preparations held at 37°C on the DMIRE2 inverted scope. Excitation illumination was generated by a 405-nm laser, and simultaneous emission was detected at 453505 nm and 525600 nm for enhanced cyan fluorescent protein (ECFP; FRET donor) and citrine (FRET acceptor), respectively. Image stacks were reconstructed with Image J software (National Institutes of Health, Bethesda, MD).
ER Ca2+ measurements. We measured biosensor function using real-time spinning disk optical confocal microscopy. Islets were placed into a microperifusion chamber mounted on an inverted epifluorescence microscope (TE-2000U, Nikon) equipped with a CARV spinning disk confocal system (Atto). Individual islets were visualized with a x20 or x40 fluorescent objective. Biosensor fluorescence excitation light was 440 nm and attenuated 5090% using neutral density filters. Emitted fluorescence at 535 nm (citrine, FRET acceptor) and 485 nm (ECFP, FRET donor) was measured using a computer-controlled high-speed filter wheel (Lambda 10-2 Optical Filter Changer, Sutter Instruments, Novato, CA); the time for changing emission filters was 60 ms. Images (100- to 250-ms exposure) were captured with a 16-bit Cascade 650 digital camera (Roper Instruments) at 10-s intervals. Imaging data acquisition and analysis were accomplished using MetaMorph/MetaFluor software (Universal Imaging). Data were expressed as background subtracted intensities of the FRET acceptor and donor fluorophores and ratio of the FRET acceptor to FRET donor emission (Ratio 535/485). In addition, data were normalized to the average baseline value of Ratio 535/485 (Relative Ratio) to facilitate comparisons between responses of different cells. In all experiments, islets were superfused with warmed (37°C) buffered salt solutions consisting of (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 1 KH2PO4, 25 NaHCO3 or 10 HEPES-NaOH (pH 7.40), and 220 glucose.
Statistical analysis. Comparisons between groups were analyzed by ANOVA (StatView Software, SAS Institute, Cary, NC), and differences were considered to be significant at P < 0.05.
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RESULTS |
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The MIP-YC3.3-er mice developed normally. Body weight and blood glucose levels of 6- and 8-wk-old transgenic animals were not significantly different from age-matched CD-1 control mice (P > 0.05; n = 6 mice from each group). The average ± SE body weight of 6-wk-old mice was 19.6 ± 2.0 g and 20.5 ± 1.8 g in the transgenic and wild-type mice, respectively. The average ± SE fasting blood glucose concentration in transgenic and wild-type mice was 158.0 ± 9.1 mg/dl and 173.0 ± 4.9 mg/dl, respectively. At 8 wk, the average ± SE fasting blood glucose in the transgenic mice was 166.6 ± 11.2 mg/dl. Intraperitoneal glucose tolerance tests performed in 6-wk-old (Fig. 1D) and 8-wk-old (data not shown) mice also demonstrated no significant differences (P > 0.05).
We used laser scanning confocal microscopy to illustrate the expression of the biosensor in paraffin-embedded pancreas sections (Fig. 2) and in single intact live islets of Langerhans (Fig. 3) from MIP-YC3.3-er mice. Immunofluorescence confocal microscopy revealed overlap between the ER Ca2+ indicator and insulin immunoreactivity (Fig. 2, AC). There was no evidence of MIP-YC3.3-er expression in islet -,
-, and PP cells (Fig. 2, DF). These results suggest that MIP-YC3.3-er is expressed only in the insulin-secreting
-cells. Double immunostaining showed that the subcellular distribution of MIP-YC3.3-er in
-cells (Fig. 2G) colocalized with GRP94, a marker of ER (Fig. 2, HI). This finding indicates that the transgenic biosensor is expressed within the lumen of the ER.
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Our confocal imaging studies demonstrated intercellular and intracellular heterogeneity of the FRET emission intensity ratio in unstimulated islets (Fig. 3D). In some cells, the perinuclear ER appeared to exhibit a higher FRET ratio than the ER in the cytoplasm (Fig. 3D, arrow). These observations raise the possibility that the FRET ratio heterogeneity might reflect regional variations in ER Ca2+ levels and suggest that -cell ER Ca2+ is regulated by different mechanisms in distinct subcellular regions. More work will need to be done to explore these hypotheses.
We next determined whether the transgenic biosensor was functional. Cameleon indicators rely on Ca2+-dependent FRET between chromophores of two GFP mutants as the basis for optical measurements of Ca2+ gradients (1, 6, 13, 17). The GFP mutants in YC3.3-er are ECFP and citrine, a mutant of enhanced yellow fluorescent protein (6). As Ca2+ concentration ([Ca2+]) rises, FRET between ECFP and citrine increases, causing fluorescence emission from ECFP to decrease and citrine to increase. In contrast, a fall in [Ca2+] reduces FRET, and consequently ECFP emission intensity increases and citrine decreases. The ratio of citrine (FRET acceptor) and ECFP (FRET donor) fluorescence emission is indicative of [Ca2+]. We measured FRET between ECFP and citrine in individual -cells within intact transgenic islets visualized (at 10-s intervals) with a real-time spinning disk confocal microscope. Approximately 5060 s after application of thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPases that causes irreversible depletion of Ca2+ sequestered within the ER (18), biosensor FRET (Fig. 4A) and the ratio of FRET acceptor-to-donor emission decreased (Fig. 4B). This effect was observed in every cell (n = 20) we examined within transgenic islets expressing YC3.3-er (Fig. 4C) and indicated that MIP-YC3.3-er was providing a readout of changes in ER [Ca2+] ([Ca2+]er). This conclusion was further substantiated by exposing islets to carbachol, a muscarinic agonist that discharges ER Ca2+ stores by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (IP3) and activation of IP3 receptor Ca2+ channels located in the ER membrane. Administration of carbachol transiently lowered [Ca2+]er, but not to the same extent as thapsigargin (Fig. 4D). The nadir of the carbachol-induced decrease in [Ca2+]er occurred within 1 min and then ER Ca2+ increased and, after 56 min, returned to baseline levels. This is consistent with ER store refilling and likely reflects activation of thapsigargin-sensitive sarco(endo)plasmic reticulum Ca2+-ATPases. However, we were surprised to observe that after depletion of ER Ca2+ stores by prolonged exposure of islets to thapsigargin (5 µM for 8 min) in solutions containing 2 mM glucose, administration of 20 mM glucose increased the average FRET ratio by 79% (n = 6 cells). This unexpected finding suggests that primary mouse
-cells possess a glucose-stimulated, thapsigargin-insensitive mechanism capable of refilling ER Ca2+ stores. The underlying mechanism remains to be determined.
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DISCUSSION |
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The reasons for the difficulty in development of mammalian transgenic biosensor models are unclear. It has been proposed that a high level of expression is necessary for the detection of cameleon fluorescence and that biosensor overexpression interferes with endogenous calmodulin-dependent signal transduction (17). On the other hand, interactions between yellow cameleon biosensors and endogenous calmodulin or calmodulin-binding proteins are unlikely; the major effect of overexpression is to increase Ca2+ buffering (12). An intracellular concentration of 20 µM is sufficient to detect cameleon fluorescence, and in vitro studies suggest that as much as 1 mM cameleon does not perturb calmodulin-dependent signaling (12). An alternate explanation for the failure of transgenic expression of protein-based Ca2+ biosensors in mammals might be the strategy employed to create the transgenic model. Our initial efforts to generate transgenic Ca2+ biosensor mice using a pancellular approach, driving transgene expression by -actin or cytomegalovirus promoters, failed. Attempts to produce transgenic YC2.1 mice, a cameleon that measures cytoplasmic Ca2+, resulted in embryonic lethality or insufficient levels of expression for detection (M. W. Roe and M. Rincon, unpublished observations). Tissue-specific targeting of YC3.3er resulted in the generation of transgenic mice with brightly fluorescent cells that were easily visualized (Fig. 1, B and C). Our studies indicate that mice tolerate expression of a GFP/calmodulin-based Ca2+ biosensor throughout maturation from neonate to adult without impairing the physiology of the transgenically targeted cells. The absence of a detectible effect of the MIP-YC3.3-er transgene on whole animal carbohydrate regulation is similar to transgenic mice expressing GFP in
-cells (7). The primary physiological function of
-cells is blood glucose sensing and insulin secretion, and it is well known that these processes are critically dependent on precise regulation of intracellular Ca2+ signaling (8). The findings suggest that MIP-YC3.3-er expression levels in the transgenic mice do not affect endogenous calmodulin-dependent physiological processes in vivo. This hypothesis is in close agreement with in vitro evidence from previous studies of the cameleons (12).
In the present study, we have established that a genetically encoded functional fluorescent Ca2+ biosensor can be expressed transgenically in intact mammalian tissues and engineered to label specific cells within a multicellular organ system. Our results suggest that cell-specific targeting of GFP-based biosensors is a feasible approach to generate transgenic expression in mammals. It is conceivable that this approach could be adapted to exploit new biosynthetic fluorescent sensor designs as they become available and advance real-time optical analysis of signal transduction and cellular biochemistry in complex organ systems of living animals.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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