The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification ...of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.
Large strain construction programs and functional analysis studies are becoming commonplace in Saccharomyces cerevisiae and involve construction of strains that carry multiple selectable marker ...genes. Extensive strain engineering is, however, severely hampered by the limited number of recyclable marker genes and by the reduced genome stability that occurs upon repeated use of heterologous recombinase‐based marker removal methods. The present study proposes an efficient method to recycle multiple markers in S. cerevisiae simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection–excision. This method relies on artificial generation of double‐strand breaks around the selection marker cassette by the meganuclease I‐SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%), suggesting that I‐SceI‐based marker removal has the potential to co‐excise an even larger number of markers. This locus‐ and marker‐independent method can be used for both dominant and auxotrophy‐complementing marker genes. Seven pDS plasmids carrying various selectable markers, which can be used for PCR‐based generation of deletion cassettes suited for I‐SceI marker recycling, are described and made available to the scientific community.
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of ...human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
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•A human organoid biobank combines hormone labeling and enteroendocrine cell generation•Transcriptomic profiling of human enteroendocrine cells uncovers differences with mice•Functional validation of EEC receptors and transcription factors•Secretome analysis reveals the repertoire of enteroendocrine secreted products
An organoid-based platform for studying human enteroendocrine cells, which sense intestinal content and release hormones to regulate many processes throughout the body, is developed by Beumer et al. and used to describe the landscape of mRNA expression and secreted products.
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