@article{SagollaLoehmannsroebenHille2013, author = {Sagolla, Kristina and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hille, Carsten}, title = {Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells}, series = {Analytical \& bioanalytical chemistry}, volume = {405}, journal = {Analytical \& bioanalytical chemistry}, number = {26}, publisher = {Springer}, address = {Heidelberg}, issn = {1618-2642}, doi = {10.1007/s00216-013-7290-6}, pages = {8525 -- 8537}, year = {2013}, abstract = {Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+](i)) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+](i) is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+](i) recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.}, language = {en} } @article{MareljaChowdhuryDoscheetal.2013, author = {Marelja, Zvonimir and Chowdhury, Mita Mullick and Dosche, Carsten and Hille, Carsten and Baumann, Otto and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Leimk{\"u}hler, Silke}, title = {The L-cysteine desulfurase NFS1 is localized in the cytosol where it provides the sulfur for molybdenum cofactor biosynthesis in humans}, series = {PLoS one}, volume = {8}, journal = {PLoS one}, number = {4}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0060869}, pages = {13}, year = {2013}, abstract = {In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.}, language = {en} } @article{ReinZimmermannHilleetal.2006, author = {Rein, Julia and Zimmermann, Bernhard and Hille, Carsten and Lang, Ingo and Walz, Bernd and Baumann, Otto}, title = {Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina}, issn = {0022-0949}, doi = {10.1242/Jeb.02187}, year = {2006}, abstract = {Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+- ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N- hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface}, language = {en} } @article{HilleWalz2006, author = {Hille, Carsten and Walz, Bernd}, title = {Dopamine-induced graded intracellular Ca2+ elevation via the Na+-Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts}, issn = {0143-4160}, doi = {10.1016/j.ceca.2005.11.006}, year = {2006}, abstract = {Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca2+ concentration ([Ca2+](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca2+ influx from the extracellular space. Additionally, dopamine induces a massive [Na+](i) elevation via the Na+- K+-2Cl(-) cotransporter (NKCC). We have reasoned that Ca2+-entry is mediated by the Na+-Ca2+ exchanger (NCE) operating in the Ca2+-entry mode. To test this hypothesis, [Ca2+](i) and [Na+](i) were measured by using the fluorescent dyes Fura- 2, Fluo-3, and SBFI. Inhibition of Na+-entry from the extracellular space by removal of extracellular Na+ or inhibition of the NKCC by 10 mu M bumetanide did not influence resting [Ca2+]i but completely abolished the dopamine-induced [Ca2+](i) elevation. Simultaneous recordings of [Ca2+](i) and [Na+](i) revealed that the dopamine-induced [Na+](i) elevation preceded the [Ca2+](i) elevation. During dopamine stimulation, the generation of an outward Na+ concentration gradient by removal of extracellular Na+ boosted the [Ca2+](i) elevation. Furthermore, prolonging the dopamine-induced [Na+](i) rise by blocking the Na+/K+-ATPase reduced the recovery from [Ca2+](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na+](i), which reverses the NCE activity into the reverse mode causing a graded [Ca2+](i) elevation in the duct cells.}, language = {en} } @phdthesis{Hille2006, author = {Hille, Carsten}, title = {Charakterisierung von Transportmechanismen in der Speicheldr{\"u}se der Schabe Periplaneta americana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-9422}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldr{\"u}se der Schabe Periplaneta americana durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldr{\"u}se sezernieren einen Prim{\"a}rspeichel, der in den Ausf{\"u}hrg{\"a}ngen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Fl{\"u}ssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt. Mikrofluorometrische Ca2+-, Na+- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivit{\"a}ten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgef{\"u}hrt. Sie sollten Informationen {\"u}ber die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. Wesentliche Ergebnisse dieser Arbeit waren: Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldr{\"u}sen in Insekten bei und erweiterte das l{\"u}ckenhafte Wissen {\"u}ber die zellul{\"a}ren Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldr{\"u}sen in Vertebraten deutlich.}, subject = {Speicheldr{\"u}se}, language = {de} } @article{RoderHille2014, author = {Roder, Phillip and Hille, Carsten}, title = {ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy}, series = {Photochemical \& photobiological sciences}, volume = {13}, journal = {Photochemical \& photobiological sciences}, number = {12}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1474-905X}, doi = {10.1039/c4pp00061g}, pages = {1699 -- 1710}, year = {2014}, abstract = {Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+](i) recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+](i) rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl-cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.}, language = {en} } @article{MutigKahlSaritasetal.2011, author = {Mutig, Kerim and Kahl, Thomas and Saritas, Turgay and Godes, Michael and Persson, Pontus and Bates, James and Raffi, Hajamohideen and Rampoldi, Luca and Uchida, Shinichi and Hille, Carsten and Dosche, Carsten and Kumar, Satish and Castaneda-Bueno, Maria and Gamba, Gerardo and Bachmann, Sebastian}, title = {Activation of the Bumetanide-sensitive Na+, K+,2Cl(-) Cotransporter (NKCC2) Is Facilitated by Tamm-Horsfall Protein in a Chloride-sensitive Manner}, series = {The journal of biological chemistry}, volume = {286}, journal = {The journal of biological chemistry}, number = {34}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M111.222968}, pages = {30200 -- 30210}, year = {2011}, abstract = {Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na+, K+,2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP-/-) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47\% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49\% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38\%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112\% versus +235\%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP-/- mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.}, language = {en} }