Susan M. Knobel

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Reduction in Pancreatic Transcription Factor PDX-1 Impairs Glucose-stimulated Insulin Secretion

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1+/− mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1+/−mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1+/− mice. The in situ perfused pancreas of PDX-1+/− mice secreted about 45% less insulin when stimulated with 16.7 mm glucose. The Km for insulin release was similar in wild type and PDX-1+/− mice. Insulin secretion in response to 20 mm arginine was unchanged; the response to 10 nm glucagon-like peptide-1 was slightly increased. However, insulin secretory responses to 10 mm 2-ketoisocaproate and 20 mm KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca2+) and that PDX-1 is important for normal function of adult pancreatic islets.

Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines.

Pancreatic β cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on β cells and results in β cell dysfunction and ultimately β cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to β cells, we used two model systems where glucose metabolism was increased in β cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3–20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in β cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in β cells. The level of β cell GK may also be critical in this process.

Quantitative imaging of the green fluorescent protein (GFP).

Publisher Summary This chapter discusses the properties of green fluorescent protein (GFP) that are important for quantitative imaging. Spectral and physical properties of GFP affect the accuracy and usefulness of any quantitative measurement. Many of these properties, such as extinction coefficient, quantum yield, photobleaching rate, and pH dependence, can be measured with purified GFP in vitro. However, other important properties, especially the time course of chromophore formation and protein degradation in vivo , cannot be easily determined. In general, one chooses the brightest, most photostable GFP available, which may make complicated corrections for background and photobleaching unnecessary in less demanding applications. The chapter also discusses the properties of the fluorescence microscope that are important in quantitative imaging, such as microscope components (objective lenses, fluorescence filters, etc.), signal-to-noise ratio, detection linearity, and fluorophore saturation. Because of the large number of GFP mutants and the variety of potential biological applications, a comprehensive description of all possible quantitative imaging situations is not possible. Thus, most descriptions of methods for quantitative imaging will be limited to the use of fluorescein-like GFP mutants with laser scanning confocal microscopy.

Adenovirus-mediated Knockout of a Conditional Glucokinase Gene in Isolated Pancreatic Islets Reveals an Essential Role for Proximal Metabolic Coupling Events in Glucose-stimulated Insulin Secretion

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between β cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gk lox). Analysis of AdenoCre-treated islets indicated that the gk lox allele in ∼30% of islet cells was converted to a nonexpressing variant (gk del). This resulted in a heterogeneous population of β cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet β cells fromgk lox/lox mice. In AdenoCre-infected islets, approximately one-third of the β cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at ∼5 mmglucose. In contrast, the normal response cells showed a sigmoidal response with an K catS0.5 of ∼8 mm. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in β cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

Quantitative imaging of metabolism by two-photon excitation microscopy.

Publisher Summary The chapter presents a discussion on quantitative imaging of metabolism by two-photon excitation microscopy. The chapter describes the use of the naturally occurring reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] as a monitor of cellular metabolism. To image these ultraviolet (UV)-absorbing fluorophores in living cells, two-photon excitation microscopy that minimizes the photodamage associated with NAD(P)H imaging is utilized. The chapter describes the use of NAD(P)H as a metabolic indicator and discusses the two-photon excitation microscopy methods, which is used to image its activity. The chapter details the instruments that are used for these experiments, with emphasis on the important design criteria for this demanding application. Finally, the chapter presents the application of two-photon excitation imaging of NAD(P)H to assay glucose-stimulated metabolism in both pancreatic and muscle cells. Fluorescence from naturally occurring NAD(P)H can be used as an indicator of cellular respiration and therefore as an intrinsic probe to study cellular metabolism. The chapter discusses the background and concepts of two-photon excitation microscopy, applications to quantitative metabolic imaging, quantitative metabolic imaging of β cells in intact pancreatic islets, quantitative imaging of muscle metabolism and several related concepts.

Real-time Analysis of Glucose Metabolism by Microscopy

Glucose metabolism has traditionally been assayed via biochemical means. Fluorescence monitoring of NAD(P)H levels has provided a non-invasive method to assay glucose metabolism in cells and tissues. However, these measurements have traditionally been of low resolution (no subcellular information) because of limitations imposed by optical and cellular photodamage problems. The recent advent of two-photon excitation microscopy as a dependable tool for biological research has opened the possibility of real-time, high-resolution analysis of glucose metabolism in living cells. Such measurements have the potential to provide subcellular information from intact tissue that cannot be obtained by other techniques.

Biphasic changes in phospholipid hydroperoxide levels during renal ischemia/reperfusion☆

The involvement of lipid peroxidation in renal ischemia/reperfusion was explored by measuring changes in the cortical content of specific primary lipid hydroperoxides (using chemluminescent detection with HPLC) following ischemia and reperfusion and by correlating the changes in hydroperoxide content with measurements of renal blood flow. Phosphatidylcholine and phosphatidylethanolamine hydroperoxide concentrations were significantly lowered during 30 or 60 min of ischemia (to levels less than 50% of control at 60 min). Following 30 min of renal ischemia, reperfusion resulted in a rebound of phospholipid hydroperoxide tissue content to levels higher than controls. Increased phospholipid hydroperoxide formation was not, however, observed in response to reperfusion following long-term (60 min) ischemia. In separate animals it was demonstrated that following 30 min ischemia and reperfusion, renal blood flow recovers to about 65% of control in 1 h. In contrast, following 60 min ischemia and reperfusion, the renal blood flow remains more highly impaired (less than 25% recovery for periods up to 24 h). These results imply that phospholipid hydroperoxides are produced and accumulate in the kidneys under normal aerobic conditions and that lipid peroxidative activity increases during renal ischemia/reperfusion to an extent dependent on the degree of local blood perfusion.

Characterization of Involution during Sea Urchin Gastrulation Using Two-Photon Excited Photorelease and Confocal Microscopy.

: Sea urchin embryos have served as a model system for the investigation of many concepts in developmental biology. Their gastrulation consists of two stages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proliferation of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate rigorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, we developed a new method for cell tracking based on two-photon excited photorelease of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cells throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biological damage. Cellular involution into the archenteron was observed throughout primary and secondary invagination, and the larval intestine was formed by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Further, as two-photon excitation microscopy becomes accessible to many researchers, the novel techniques described here will be broadly applicable to development of other invertebrate and vertebrate embryos.

Kinetic characterization of a stably expressed novel Na+/H+ exchanger (NHE-2).

We have recently reported the molecular cloning, sequencing and tissue distribution of a novel Na+/H+ exchanger (NHE-2). The cDNA for NHE-2 was cloned by screening a rat intestinal cDNA library. This clone was unique due to the fact that it lacks the first two transmembrane domains which are present in the other Na+/H+ exchanger isoforms (NHE-1, NHE-3, NHE-4). This structural change in the cDNA offered a unique opportunity to study in detail the properties of this stably expressed cDNA in chinese lung fibroblasts that lack the Na+/H+ exchanger (PS120) cells. Amiloride-sensitive Na+ uptake was linear up to 2 min in PS120 cells transfected with the cDNA. Kinetics of the amiloride-sensitive Na+ uptake showed a Vmax of 24.7 +/- 5 nmol/microliters ICW per min and a Km of 33.1 +/- 2.0 mM. The inhibitory constant (KI) for amiloride and its analogue 5-N-ethyl-N-isopropylamiloride (EIPA) was 0.15 microM and 0.66 microM, respectively. Epidermal growth factor, a known stimulator of NHE-1, also upregulated the expressed NHE-2. These results characterize the kinetic properties of this unique exchanger and suggests that the first two transmembrane domains of the Na+/H+ exchanger isoforms are not essential for the expression of amiloride-sensitive Na+ uptake.