The Popeye domain containing (POPDC) gene family encodes a novel class of membrane-bound cyclic AMP effector proteins. POPDC proteins are abundantly expressed in cardiac and skeletal muscle. Consistent with its predominant expression in striated muscle, Popdc1 and Popdc2 null mutants in mouse and zebrafish develop cardiac arrhythmia and muscular dystrophy. Likewise, mutations in POPDC genes in patients have been associated with cardiac arrhythmia and muscular dystrophy phenotypes. A membrane trafficking function has been identified in this context. POPDC proteins have also been linked to tumour formation. Here, POPDC1 plays a role as a tumour suppressor by limiting c-Myc and WNT signalling. Currently, a common functional link between POPDC's role in striated muscle and as a tumour suppressor is lacking. We also discuss several alternative working models to better understand POPDC protein function.

HighlightsA method for 3D reconstruction of serial 2D histology image stacks is proposed.Pre-alignment to an external pre-cut reference (blockface) prevents shape artifacts.Formulated as diffusion of transformations from each slice to its neighbors.Registrations replaced by much faster transformation operations.

The Popeye domain-containing (POPDC) gene family, consisting of Popdc1 (also known as Bves), Popdc2, and Popdc3, encodes transmembrane proteins abundantly expressed in striated muscle. POPDC proteins have recently been identified as cAMP effector proteins and have been proposed to be part of the protein network involved in cAMP signaling. However, their exact biochemical activity is presently poorly understood. Loss-of-function mutations in animal models causes abnormalities in skeletal muscle regeneration, conduction, and heart rate adaptation after stress. Likewise, patients carrying missense or nonsense mutations in POPDC genes have been associated with cardiac arrhythmias and limb-girdle muscular dystrophy. In this review, we introduce the POPDC protein family, and describe their structure function, and role in cAMP signaling. Furthermore, the pathological phenotypes observed in zebrafish and mouse models and the clinical and molecular pathologies in patients carrying POPDC mutations are described.

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-S H74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.FRET requires sufficient overlap of excitation spectrum of the acceptor with the emission spectrum of the donor and also depends on the relative angular orientations of the donor and acceptor dipoles [4].In the case of heteroFRET, the two fluorophores are not identical and emit at different wavelengths [5]. There are a very large number of approaches to analysing spectral ratio-metric FRET data [6,7], the simplest of which is a straight-forward ratio of sensitized acceptor emission to the directly excited emission from the donor fluorophore. This simple calculation of the FRET ratio is useful for real-time monitoring of biosensor readouts in live cells and tissue slices.Over recent years, a number of non-proprietary software packages have been written for ratio-metric FRET analysis, however their use is limited to analysis of previously saved image-stacks [8][9][10][11]. Some proprietary software offers real-time measurements of the ratios between fluorescent intensities in two channels; one example is MetaFluor (Molecular Devices, San Jose, USA) [12]. This provides additional flexibility, allowing experimental procedures to be carried out in response to changes in the FRET ratio. As a low-cost alternative, Julia U. Sprenger et al. (2012) built a customised epifluorescence FRET imaging system and developed a non-proprietary ImageJ macro, which displays real-time ratio-metric data obtained from a Micro-Manager controlled time-lapse acquis...

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