Suihan Feng

Synthesis of a BODIPY Library and Its Application to the Development of Live Cell Glucagon Imaging Probe

The first BODIPY library (BD) was synthesized, and a highly selective glucagon sensor, Glucagon Yellow (BD-105), was discovered by fluorescence image-based screening method. BD library was synthesized via a Knoevenagel-type condensation reaction with 160 benzaldehydes and the 1,3 dimethyl-BODIPY scaffold. Using BD compounds, a fluorescence image-based screening was performed against three cell lines including AlphaTC1 and BetaTC6 cells which secret glucagon and insulin, respectively, and HeLa as control cells. Out of the 160 candidate probes, one compound, Glucagon Yellow, exhibited selective staining only in AlphaTC1 cells. The selectivity of Glucagon Yellow toward glucagon was confirmed in vitro by comparison of its fluorescence intensity change against 19 biologically relevant analytes. Subsequent immunostaining experiments revealed that Glucagon Yellow and the glucagon antibody colocalized in pancreas tissue, showing a high quantitative correlation analysis by the Pearsons coefficient constant (R(r) = 0.950). These results demonstrated the potential application of Glucagon Yellow as a glucagon imaging agent in live cells and tissues.

Recapture of GFP Chromophore Fluorescence in a Protein Host

When encapsulated by human serum albumin (HSA), certain derivatives of the green fluorescent protein (GFP) chromophore recover their fluorescence due to inhibition of torsional motion. These derivatives show remarkable sensitivity and selectivity as well as favorable spectroscopic properties toward HSA, thus providing selective probes for this and similar proteins and demonstrating the use of GFP chromophores as topological fluorophores.

The fatty acid composition of diacylglycerols determines local signaling patterns.

Cellular signals are transduced through vast networks of proteins and small molecule metabolites. Rigorous control of the respective signaling molecules is required to ensure a precise and reproducible outcome. On the protein level this is often accomplished by specific reversible chemical modifications such as phosphorylation or by localization of proteins to defined cellular compartments. 2] Much less is known about cellular mechanisms that control small-molecule-mediated signaling events. This is largely due to the intrinsically more difficult observation of small-molecule turnover and localization in living cells. These difficulties are potentiated when lipid signaling is investigated. The variety of known lipid backbones is fairly comprehensive but the diversity and combinations of fatty acids attached to these backbones provides many thousand possibilities and lipidomics shows that a large portion of this diversity is available in cells. This overwhelming and generally not addressable complexity has led to a situation where lipid signaling events are treated as head-group signaling events and the existing chemical differences between individual species of the same lipid class are widely ignored although a number of in vitro studies suggest significant differences in potency. Along the same lines, the influence of subcellular concentration gradients of defined lipid species on intracellular signaling has not been studied thoroughly so far. We hypothesized that both lipid species diversity and subcellular concentration gradients of distinct lipid species might serve as molecular mechanisms to drive specific lipid-mediated signaling events. Experimentally, both fatty acid diversity and locally elevated levels of a given species may be generated by using photoactivatable lipids in intact cells. We chose to analyze diacylglycerol (DAG) signaling due to its important role in several cellular signaling pathways that include G-protein coupled receptors as well as growth factor triggered and calcium-based signaling networks. Recent lipidomics analyses demonstrated the co-existence of 30–50 DAG species with different fatty acid compositions in mammalian cells. While DAGs are best known to activate various protein kinase C (PKC) isoforms by binding to their C1 domains and recruiting them to cellular membranes, DAG-induced translocation and activation of proteins such as RasGRPs, Munc13, and DGKg have also been described. In addition, DAGs have been shown to directly activate human transient receptor potential C3 (TRPC3) and TRPC6 channels. So far, locally elevated DAG levels have either been experimentally achieved by liberation of a photoactivatable T-cell receptor agonist which causes downstream DAG production or by local uncaging of a nonphysiological DAG analogue. 23] While these approaches have led to important insights into the mechanism of T-cell receptor mediated signaling and microtubule-organizing center (MTOC) polarization in T-cells, they cannot be utilized

Fluorescence response profiling for small molecule sensors utilizing the green fluorescent protein chromophore and its derivatives.

Using a fluorescence response profile, a systematic examination was performed for synthetic chromophores of the green fluorescent protein (GFP) to discover new small molecule sensors. A group of 41 benzylideneimidazolinone compounds (BDI) was prepared and screened toward 94 biologically relevant analytes to generate fluorescence response profiles. From the response pattern, compounds containing aminobenzyl and heteroaromatic cyclic substructures revealed a pH dependent emission decrease effect, and unlike other fluorescence scaffolds, most BDIs showed fluorescence quenching when mixed with proteins. On the basis of the primary response profile, we obtained three selective fluorescence turn-on sensors for pH, human serum albumin (HSA), and total ribonucleic acid (RNA). Following analysis, a fluorescence response profile testing four nucleic acids revealed the alkyloxy (Ph-OR) functional group in the para position of benzyl analogues contributes to RNA selectivity. Among the primary hit compounds, BDI 2 showed outstanding selectivity toward total RNA with 5-fold emission enhancement. Finally, BDI 24 showed selective fluorescence increase to HSA (K(d) = 3.57 μM) with a blue-shifted emission max wavelength (Δλ(em) = 15 nm). These examples of fluorescence sensor discovery by large-scale fluorescence response profiling demonstrate the general applicability of this approach and the usefulness of the response profiles.

A rapidly reversible chemical dimerizer system to study lipid signaling in living cells

Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

Exclusive photorelease of signalling lipids at the plasma membrane

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Reversible chemical dimerizer-induced recovery of PIP2 levels moves clathrin to the plasma membrane

Chemical dimerizers are powerful non-invasive tools for bringing molecules together inside intact cells. We recently introduced a rapidly reversible chemical dimerizer system which enables transient translocation of enzymes to and from the plasma membrane (PM). Here we have applied this system to transiently activate phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown at the PM via translocation of phosphoinositide 5-phosphatase (5Ptase). We found that the PIP2 sensor phospholipase C-δ PH domain (PLCδ-PH) is released from the PM upon addition of the reversible chemical dimerizer rCD1. By outcompeting rCD1, rapid release of the 5Ptase from the PM is followed by PIP2 recovery. This permits the observation of the PIP2-dependent clathrin assembly at the PM.

T-CrAsH: a heterologous chemical crosslinker.

Copper‐free click chemistry is currently the most promising and most rapidly developing technology for performing tailored chemical reactions inside intact living cells and animals. Its potential is particularly intensely explored in the field of live cell imaging, for both proteins and metabolites. Here we expand the application spectrum of click reactions to the chemical crosslinking of two proteins of choice in living cells. By combining strain‐promoted Diels–Alder cycloaddition with FlAsH‐based labeling of peptidic tetracysteine motifs, we developed the membrane‐permeating reversible crosslinker T‐CrAsH. We demonstrate the feasibility of the method both in vitro and inside cells. The biggest advantage of this new tool is the small size of the crosslinkable groups; this significantly decreases the risk of functional interference.

A single-cell model of PIP3 dynamics using chemical dimerization.

Most cellular processes are driven by simple biochemical mechanisms such as protein and lipid phosphorylation, but the sum of all these conversions is exceedingly complex. Hence, intuition alone is not enough to discern the underlying mechanisms in the light of experimental data. Toward this end, mathematical models provide a conceptual and numerical framework to formally evaluate the plausibility of biochemical processes. To illustrate the use of these models, here we built a mechanistic computational model of PI3K (phosphatidylinositol 3-kinase) activity, to determine the kinetics of lipid metabolizing enzymes in single cells. The model is trained to data generated upon perturbation with a reversible small-molecule based chemical dimerization system that allows for the very rapid manipulation of the PIP3 (phosphatidylinositol 3,4,5-trisphosphate) signaling pathway, and monitored with live-cell microscopy. We find that the rapid relaxation system used in this work decreased the uncertainty of estimating kinetic parameters compared to methods based on in vitro assays. We also examined the use of Bayesian parameter inference and how the use of such a probabilistic method gives information on the kinetics of PI3K and PTEN activity.

Accelerating fluorescent sensor discovery

Herein, we report the first systematic and unbiased evaluation of the BODIPY fluorophore library against a wide panel of biologically relevant molecules, and discoveries of 2 novel fluorescent probes for BSA and dopamine.