Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p

The Neurokinin 1 Receptor Antagonist, Ezlopitant, Reduces Appetitive Responding for Sucrose and Ethanol

Abstract Background: The current obesity epidemic is thought to be partly driven by over-consumption of sugar-sweetened diets and soft drinks. Loss-of-control over eating and addiction to drugs of abuse share overlapping brain mechanisms including changes in motivational drive, such that stimuli that are often no longer ‘liked’ are still intensely ‘wanted’ [7,8]. The neurokinin 1 (NK1) receptor system has been implicated in both learned appetitive behaviors and addiction to alcohol and opioids; however, its role in natural reward seeking remains unknown. Methodology/Principal Findings: We sought to determine whether the NK1-receptor system plays a role in the reinforcing properties of sucrose using a novel selective and clinically safe NK1-receptor antagonist, ezlopitant (CJ-11,974), in three animal models of sucrose consumption and seeking. Furthermore, we compared the effect of ezlopitant on ethanol consumption and seeking in rodents. The NK1-receptor antagonist, ezlopitant decreased appetitive responding for sucrose more potently than for ethanol using an operant self-administration protocol without affecting general locomotor activity. To further evaluate the selectivity of the NK1-receptor antagonist in decreasing consumption of sweetened solutions, we compared the effects of ezlopitant on water, saccharin-, and sodium chloride (NaCl) solution consumption. Ezlopitant decreased intake of saccharin but had no effect on water or salty solution consumption. Conclusions/Significance: The present study indicates that the NK1-receptor may be a part of a common pathway regulating the self-administration, motivational and reinforcing aspects of sweetened solutions, regardless of caloric value, and those of substances of abuse. Additionally, these results indicate that the NK1-receptor system may serve as a therapeutic target for obesity induced by over-consumption of natural reinforcers

Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development

Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (mu-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish brown locus homologues tyrp1a/b eliminated accumulation of these elements in the RPE, indicating that they are bound by mature melanosomes. Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.Peer reviewe

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions