Biology subjects

Griesbeck, O.

Publications and source records attributed to Griesbeck, O..

5 recordsLinked to original sources

bioRxiv2026

A red fluorescent sensor for intensity-and lifetime imaging of cAMP in living cells

cAMP (cyclic adenosine monophosphate) is a crucial intracellular messenger involved in neuromodulation and plasticity, but more information on its spatiotemporal regulation is desirable. Here we constructed a red fluorescent indicator based on a truncated cAMP binding site derived from a regulatory PKA subunit of protozoan origin. It contains a single binding site for cAMP highly specific for cAMP over cGMP. chILI (cAMP highly-responsive Intensity and Lifetime Indicator) can be imaged as an intensity-based probe, but alternatively also offers options for fluorescence lifetime imaging. When expressed in the mouse brain using AAV-vector mediated gene transfer, it reports spontaneous and evoked cAMP transients from CA1 neurons in the hippocampus of awake mice. chILI will add valuable options for monitoring cAMP in vitro and in vivo.

neuroscience↗

bioRxiv2026

EverGreen: A Calcium Sensing Fluorescent Protein with Ultra-Slow Response Properties

Fluorescent calcium indicators are typically optimized for fast kinetics, but for some bio-engineering projects in synthetic biology slower indicators with long decay time constants may be more practical and easier to apply. We here present a green fluorescent protein calcium biosensor with unusually slow response kinetics. By molecular engineering we were able to extend a slow component of the decay time to 6.1 min. The fluorescence in EverGreen could thus be detected for minutes after the underlying calcium fluctuation had already dissipated. This may be useful when constraints in instrumentation and methodology do not allow close coupling of physiological activation of cells and read-out of signals, facilitating directed evolution projects run via plate readers or cell sorters.

bioengineering↗

bioRxiv2023

Fluorescent Sensors for Imaging Interstitial Calcium

Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the lack of methods that allow monitoring in tight inter-cell spaces at high spatio-temporal resolution. We engineered high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (KD) ranging from 0.8 mM to 2.9 mM, making them uniquely tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.

physiology↗

bioRxiv2022

Calcium flux through ER-TGN contact sites facilitates cargo export

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon a local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains elusive, as the cytosol has a very low steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), whereby the close apposition of the two organelles allows the protein-mediated exchange of molecular species such as lipids. Here we show that the TGN export of Cab45 clients requires the integrity of ER-TGN MCS and IP3R-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real-time. These data show for the first time that ER-TGN MCS facilitates Ca2+ transfer required for SPCA1-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology. SummaryThe current study demonstrates that the trafficking of COMP and LyzC relies on Ca2+ flux between the endoplasmic reticulum (ER) and trans-Golgi Network (TGN). This process requires the activity of IP3 receptors, present in ER membranes, and depends on the integrity of the membrane contact site between these two organelles.

cell biology↗

bioRxiv2019

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles

Summary sentenceBoth FSH and LH increase Ca2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors.\n\nABSTRACTIn mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ~10 minutes, remains above baseline for another ~10 minutes, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 hours and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does slightly attenuate ovulation. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

cell biology↗