Conformational analysis of a genetically encoded FRET biosensor by SAXS.

Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.

Annexin A4 self-association modulates general membrane protein mobility in living cells.

Annexins are Ca2+-regulated phospholipid-binding proteins whose function is only partially understood. Annexin A4 is a member of this family that is believed to be involved in exocytosis and regulation of epithelial Cl- secretion. In this work, fluorescent protein fusions of annexin A4 were used to investigate Ca2+-induced annexin A4 translocation and self-association on membrane surfaces in living cells. We designed a novel, genetically encoded, FRET sensor (CYNEX4) that allowed for easy quantification of translocation and self-association. Mobility of annexin A4 on membrane surfaces was investigated by FRAP. The experiments revealed the immobile nature of annexin A4 aggregates on membrane surfaces, which in turn strongly reduced the mobility of transmembrane and plasma membrane associated proteins. Our work provides mechanistic insight into how annexin A4 may regulate plasma membrane protein function.

FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events.

The number of fluorescent sensors and their use in living cells has significantly increased in the past years. Yet, the analysis of data from single cells or cell populations usually remains a very time-consuming enterprise. Here, we introduce FluoQ, a new macro for the image analysis software ImageJ, which enables fast analysis of multiparameter time-lapse fluorescence microscopy data with minimal manual input. FluoQ provides statistical analysis of all measured parameters and delivers the results in multiple graphic and numeric displays. We demonstrate the power of FluoQ by applying the macro to data analysis in the development and optimization of novel FRET reporters for monitoring the performance of calcium/calmodulin-binding inositol trisphosphate kinases A and B (ITPKA and ITPKB) in HeLa cells. We find that conformational changes in the ITPKA-based sensor follow receptor-mediated calcium oscillations. This indicates that ITPKA contributes to the regulation of intracellular calcium transients by limiting inositol trisphosphate levels.

Rapid development of genetically encoded FRET reporters.

To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.

Simultaneous recording of multiple cellular events by FRET.

The function of many sensors for measuring intracellular events is based on Förster resonance energy transfer (FRET). Here we demonstrate for the first time the use of multiple ratiometric FRET sensors in parallel through spatial and spectral resolution. We monitored three calcium-dependent signaling events by a cytosolic sensor for calcium/calmodulin-dependent protein kinase IIalpha, a membrane-bound sensor for protein kinase C, and a translocating FRET probe based on annexin A4. This multiparameter imaging approach gives insight into the precise timing of cellular events within one single cell, thereby providing a major advantage over single-parameter protocols. This type of imaging will likely be important for high content cell analysis and screening efforts in the future.

Heterogeneity and timing of translocation and membrane-mediated assembly of different annexins.

Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.