"Radical" differences between two FLIM microscopes affect interpretation of cell signaling dynamics.

The outcome of cell signaling depends not only on signal strength but also on temporal progression. We use Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to investigate intracellular signaling dynamics. We examined the ß1 receptor-Gαs-cAMP signaling axis using both widefield frequency domain FLIM (fdFLIM) and fast confocal time-correlated single photon counting (TCSPC) setups. Unexpectedly, we observed that fdFLIM revealed transient cAMP responses in HeLa and Cos7 cells, contrasting with sustained responses as detected with TCSPC. Investigation revealed no light-induced effects on cAMP generation or breakdown. Rather, folic acid present in the imaging medium appeared to be the culprit, as its excitation with blue light sensitized degradation of ß1 agonists. Our findings highlight the impact of subtle phototoxicity on experimental outcomes, advocating confocal TCSPC for reliable analysis of response kinetics and stressing the need for full disclosure of chemical formulations by scientific vendors.

Dynamic FRET-FLIM based screening of signal transduction pathways.

Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein-protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-SH189. Many hundreds of cells were recorded at 5 s intervals for each condition. FLIM time traces were calculated for every cell, and decay kinetics were obtained. cAMP clearance was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. However, taking advantage of the quantitative FLIM data, we found that knockdown of individual PDEs has a very limited effect on baseline cAMP levels. By combining photon-efficient FLIM instrumentation with optimized sensors, systematic gene knockdown and an automated open-source analysis pipeline, our study demonstrates that dynamic screening of transient cell signals has become feasible. The quantitative platform described here provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

DINAX- a comprehensive database of inherited ataxias.

BACKGROUND: Neurodegenerative disorders such as hereditary ataxia often manifest overlapping symptoms and are likely to be misdiagnosed based on clinical phenotypes. To identify the genes associated with such disorders for diagnostic purposes, geneticists often use high throughput technologies which generate an enormous amount of data on variants whose relevance can be unclear. Besides, analysis and interpretation of high throughput data require gleaning of several web-based resources which can be laborious and time-consuming. To overcome these, we have created a Database for Inherited Ataxia (DINAX), a repository of gene variants from publicly available information. METHODS: DINAX is implemented as a MySQL relational database using the PHP scripting language. Web interfaces were developed using HTML, CSS, and JavaScript. Variant and phenotype information was collected and manually curated from published literature and primary databases such as OMIM and ClinVar. These were further analyzed to decipher expression and pathway analysis. RESULTS: DINAX is an inventory of 7166 genomic variants (single nucleotide polymorphisms, deletions, insertions, and translocations) reported till date among the 185 genes associated with different subtypes of inherited ataxia. DINAX implements a dual search methodology for genes and phenotypes linking to ataxia associated genes, variants, and their source. Pathway analysis confirmed their association with ataxia. CONCLUSION: The database is created to provide a single web source for obtaining information about ataxia related genes. Besides, the database facilitates easy identification of known and reported variants as well as the novel or unreported variants. DINAX is freely available at http://slsdb.manipal.edu/dinax.