Cytosolic-enhanced dark Epac-based FRET sensors allow for intracellular cAMP detection in live cells via FLIM.

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for studying second messengers with high temporal and spatial resolution. FRET is commonly detected by ratio imaging, but fluorescence lifetime imaging microscopy (FLIM), which measures the donor fluorophore's lifetime, offers a robust and more quantitative alternative. We have introduced and optimized four generations of FRET sensors for cAMP, based on the effector molecule Epac1, including variants for either ratio imaging or FLIM detection.

"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 β1 receptor-G-cAMP signaling axis using both widefield frequency domain FLIM (fdFLIM) and fast confocal time-correlated single photon counting (TCSPC) setups.

Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors.

Second messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET.

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.

TRPM7 residue S1269 mediates cAMP dependence of Ca2+ influx.

The nonspecific divalent cation channel TRPM7 (transient receptor potential-melastatin-like 7) is involved in many Ca2+ and Mg2+-dependent cellular processes, including survival, proliferation and migration. TRPM7 expression predicts metastasis and recurrence in breast cancer and several other cancers. In cultured cells, it can induce an invasive phenotype by promoting Ca2+-mediated epithelial-mesenchymal transition.

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation.

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove.

TRPM7 controls mesenchymal features of breast cancer cells by tensional regulation of SOX4.

Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo.

Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors.

Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin.

Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity.

Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio.

Recording intracellular cAMP levels with EPAC-based FRET sensors by fluorescence lifetime imaging.

Eukaryotic cells use second messengers such as Ca(2+), IP3, cGMP, and cAMP to transduce extracellular signals like hormones, via membrane receptors to downstream cellular effectors. FRET-based sensors are ideal to visualize and measure these rapid changes of second messenger concentrations in time and place. Here, we describe the use of EPAC-based FRET sensors to measure cAMP with spatiotemporal resolution in cells by fluorescence lifetime imaging (FLIM).

The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons.

The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-S(H150)) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2.

TRPM7 triggers Ca2+ sparks and invadosome formation in neuroblastoma cells.

Cell migration depends on the dynamic formation and turnover of cell adhesions and is tightly controlled by actomyosin contractility and local Ca2+ signals. The divalent cation channel TRPM7 (Transient Receptor Potential cation channel, subfamily Melastatin, member 7) has recently received much attention as a regulator of cell adhesion, migration and (localized) Ca2+ signaling. Overexpression and knockdown of TRPM7 affects actomyosin contractility and the formation of cell adhesions such as invadosomes and focal adhesions, but the role of TRPM7-mediated Ca2+ signals herein is currently not understood.

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells.

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP.

A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.

FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM) or by Sensitized Emission (SE), e.

Fas/CD95 down-regulation in lymphoma cells through acquired alkyllysophospholipid resistance: partial role of associated sphingomyelin deficiency.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP.

Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells.

Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts.

A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells.

Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway.