FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events.
Stein, F., Kress, M., Reither, S., Piljic, A. & Schultz, C.
ACS Chem Biol. 2013 Sep 20;8(9):1862-8. doi: 10.1021/cb4003442. Epub 2013 Aug 6.
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.
The fatty acid composition of diacylglycerols determines local signaling patterns.
Nadler, A., Reither, G., Feng, S., Stein, F., Reither, S., Muller, R. & Schultz, C.
Angew Chem Int Ed Engl. 2013 Jun 10;52(24):6330-4. doi: 10.1002/anie.201301716.Epub 2013 May 29. Europe PMC
In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.
Haberkant, P., Raijmakers, R., Wildwater, M., Sachsenheimer, T., Brugger, B., Maeda, K., Houweling, M., Gavin, A.C., Schultz, C., van Meer, G., Heck, A.J. & Holthuis, J.C.
Angew Chem Int Ed Engl. 2013 Apr 2;52(14):4033-8. doi: 10.1002/anie.201210178.Epub 2013 Feb 28. Europe PMC
Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.
Posor, Y., Eichhorn-Gruenig, M., Puchkov, D., Schoneberg, J., Ullrich, A., Lampe, A., Muller, R., Zarbakhsh, S., Gulluni, F., Hirsch, E., Krauss, M., Schultz, C., Schmoranzer, J., Noe, F. & Haucke, V.
Nature. 2013 Jul 11;499(7457):233-7. doi: 10.1038/nature12360. Epub 2013 Jul 3.
Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2alpha (PI(3)K C2alpha) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2alpha impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2alpha is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.
Protein tango: the toolbox to capture interacting partners.
Rutkowska, A. & Schultz, C.
Angew Chem Int Ed Engl. 2012 Aug 13;51(33):8166-76. doi: 10.1002/anie.201201717.Epub 2012 Jun 11.
The evaluation of protein function in the context of the whole cell is crucial for understanding of living systems. In this context, the identification and modulation of protein-protein interactions in and outside cells is of ample importance. Several methods have been developed in the past years to detect and/or actively induce protein-protein interactions in living cells. As a result, tools are now available to manipulate intracellular events by reversible or irreversible cross-linking of proteins in a specific manner. These techniques open many new doors and enable the dissection of complicated protein networks. Herein we describe which cross-linkers and inducers of dimerization are out there and how to make use of this great toolbox.
Target-activated prodrugs for the auto-regulated inhibition of MMP12.
Cobos-Correa, A., Stein, F., Schultz, C.
ACS Med. Chem. Lett., 2012, 3 (8), pp 653-657, doi: 10.1021/ml3001193.
We describe a prodrug concept in which the target enzyme MMP12 produces its own inhibitor in a two-step activation procedure. By using an MMP12-specific peptide sequence and a known sulfonamide drug integrated in the backbone, the active inhibitor is released upon enzyme cleavage. In in vitro experiments, we present proof of concept that the activation proceeds with useful kinetics. The approach is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in a highly specific manner only at such sites where enzyme activity resides.
Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters.
Gehrig, S., Mall, M.A. & Schultz, C.
Angew Chem Int Ed Engl. 2012 Jun 18;51(25):6258-61. doi: 10.1002/anie.201109226.Epub 2012 May 3.
Finding NEmo: In a mouse model for lung inflammation, the lipidated peptide-based ratiometric fluorescent reporter NEmo-2 indicates that neutrophil elastase (NE) activity is mainly associated with the surface of neutrophils, whereas a soluble reporter variant showed no activity in the lung fluid, likely because of the abundance of antiproteases. Targeting the localization of NE activity has great potential for the development of improved antiinflammatory drugs.
Amino acids for diels-alder reactions in living cells.
Plass, T., Milles, S., Koehler, C., Szymanski, J., Mueller, R., Wiessler, M., Schultz, C. & Lemke, E.A.
Angew Chem Int Ed Engl. 2012 Apr 23;51(17):4166-70. doi: 10.1002/anie.201108231.Epub 2012 Mar 30.
Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site-specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans-cyclooctene units that react with tetrazine derivatives in an inverse-electron-demand Diels-Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide-cyclooctyne click reaction (right).