The fundamental theory of Förster resonance energy transfer (FRET) was established in the 1940's. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation and analytical tools. Combined with state-of-the-art microscopy and spectroscopy, FRET imaging allows scientists to study a variety of phenomena that produce changes in molecular proximity, thereby leading to many significant findings in the life sciences. In this review, we outline various FRET imaging techniques and their strengths and limitations; we also provide a biological model to demonstrate how to investigate protein-protein interactions in living cells using both intensity- and fluorescence lifetime-based FRET microscopy methods.

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ϳ20 m h ؊1 , which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.Fluorescence microscopy has revolutionized our understanding of the bacterial cell and provided new opportunities to investigate the behavior of cell division proteins and chromosome dynamics in bacteria (25). Central to this research is the application of time-lapse microscopy to study bacterial cell division, which has revealed the complexity with which bacteria coordinate cellular growth and division. For example, the rodshaped bacteria Escherichia coli and Bacillus subtilis incorporate new peptidoglycan into their cell wall along their lateral walls, while coccoid bacteria such as Staphylococcus aureus do so at mid-cell (4). Actinobacteria, such as Corynebacterium and members of the mycelial, antibiotic-producing genus Streptomyces, incorporate peptidoglycan at the cell poles (4). In the case of streptomycetes, this allows them to adopt a hyphal growth strategy through peptidoglycan incorporation at the hyphal tip (9). This is ideally suited to the colonization of their particulate habitat, the soil, through the generation of a mycelium that permits nutrients to be transported from a nutrient reservoir to the actively growing tip. As such, streptomycetes represent a group of organisms that grow in a fashion distinct from other, better understood bacteria. The knowledge base associated with morphological and physiological differentiation in the model organism Streptomy...

Exposure to hypoxia elicits an increase in minute ventilation that diminishes during continued exposure (roll-off). Brainstem Nmethyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS) contribute to the initial hypoxia-induced increases in minute ventilation. Roll-off is regulated by platelet-derived growth factor receptor-b (PDGFR-b) and S-nitrosoglutathione (GSNO) reductase (GSNOR). S-nitrosylation inhibits activities of NMDAR and nNOS, but enhances GSNOR activity. The importance of S-nitrosylation in the hypoxic ventilatory response is unknown. This study confirms that ventilatory roll-off is virtually absent in female GSNOR 1/2 and GSNO 2/2 mice, and evaluated the location of GSNOR in female mouse brainstem, and temporal changes in GSNOR activity, protein expression, and S-nitrosylation status of GSNOR, NMDAR (1, 2A, 2B), nNOS, and PDGFR-b during hypoxic challenge. GSNOR-positive neurons were present throughout the brainstem, including the nucleus tractus solitarius. Protein abundances for GSNOR, nNOS, all NMDAR subunits and PDGFR-b were not altered by hypoxia. GSNOR activity and S-nitrosylation status temporally increased with hypoxia. In addition, nNOS Snitrosylation increased with 3 and 15 minutes of hypoxia. Changes in NMDAR S-nitrosylation were detected in NMDAR 2B at 15 minutes of hypoxia. No hypoxia-induced changes in PDGFR-b Snitrosylation were detected. However, PDGFR-b phosphorylation increased in the brainstems of wild-type mice during hypoxic exposure (consistent with roll-off), whereas it did not rise in GSNOR 1/2 mice (consistent with lack of roll-off). These data suggest that: (1) S-nitrosylation events regulate hypoxic ventilatory response; (2) increases in S-nitrosylation of NMDAR 2B, nNOS, and GSNOR may contribute to ventilatory roll-off; and (3) GSNOR regulates PDGFR-b phosphorylation.Keywords: hypoxic ventilatory response; neuronal nitric oxide synthase; N-methyl-D-aspartate receptor; platelet-derived growth factor receptor-b; S-nitrosoglutathione reductase Clinical RelevanceThis article reports that the change in S-nitrosylation status of key brainstem proteins may underlie the roll-off phase of the hypoxic ventilatory response. This has potential clinical applications for understanding how mechanisms that affect S-nitrosothiol bioavailability (i.e., S-nitrosoglutathione reductase) play a role in central breathing disorders.

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