Cell Calcium Imaging Research Articles

Identification and expression profiles of gustatory receptor genes in Bactrocera minax larvae (Diptera: Tephritidae): Role of BminGR59f in larval growth.

Insects employ various types of gustatory receptors (GRs) to identify nutrient-rich food and avoid toxic substances. The larval gustatory system is the critical checkpoint for food acceptance or rejection. As a specialist herbivore, the larvae of Bactrocera minax feed only on unripe citrus fruits. However, how larvae use GRs to check and adapt to the secondary metabolites in unripe citrus fruits remains unknown. In this study, we first performed developmental expression profiles showing that most BminGRs genes were highly expressed in 1st and 2nd instar larvae and that tissue-specific expression indicated high expression of most BminGRs genes in the mouthparts of 2nd instar larvae. Furthermore, we found that silencing BminGR59f by RNA interference (RNAi) affected the growth of 2nd instar B. minax larvae. Hesperidin and naringin were screened as ligands of BminGR59f via RNAi and cell calcium imaging, and the combination of these two flavones increased the body weight of larvae. In summary, we identified a novel gustatory perception pattern in B. minax for detecting hesperidin and naringin, which boosted the growth of B. minax larvae. These results shed light on how specialist herbivores detect and adapt to host metabolites in adverse environments depending on larval GRs.

Discovery of Orphan Olfactory Receptor 6M1 as a New Anticancer Target in MCF-7 Cells by a Combination of Surface Plasmon Resonance-Based and Cell-Based Systems

Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.

Cell Calcium Imaging as a Reliable Method to Study Neuron-Glial Circuits.

Complex dynamic cellular networks have been studied in physiological and pathological processes under the light of single-cell calcium imaging (SCCI), a method that correlates functional data based on calcium shifts operated by different intracellular and extracellular mechanisms integrated with their cell phenotypes. From the classic synaptic structure to tripartite astrocytic model or the recent quadripartite microglia added ensemble, as well as other physiological tissues, it is possible to follow how cells signal spatiotemporally to cellular patterns. This methodology has been used broadly due to the universal properties of calcium as a second messenger. In general, at least two types of receptor operate through calcium permeation: a fast-acting ionotropic receptor channel and a slow-activating metabotropic receptor, added to exchangers/transporters/pumps and intracellular Ca2+ release activated by messengers. These prototypes have gained an enormous amount of information in dynamic signaling circuits. SCCI has also been used as a method to associate phenotypic markers during development and stage transitions in progenitors, stem, vascular cells, neuro- and glioblasts, neurons, astrocytes, oligodendrocytes, and microglia that operate through ion channels, transporters, and receptors. Also, cancer cells or inducible cell lines from human organoids characterized by transition stages are currently being used to model diseases or reconfigure healthy cells in terms of the expression of calcium-binding/permeable molecules and shed light on therapy.

38.1 IMPACT OF ANTI-NMDA RECEPTOR AUTOANTIBODIES FROM PSYCHOTIC PATIENTS ON THE GLUTAMATE SYNAPSE

BackgroundThe flourishing identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, their putative presence in different diseases, as well as in healthy subjects, has raised questions about detection reliability and pathogenic role.MethodsUsing a combination of single molecule-based imaging approaches, cell calcium imaging, and single-cell electrophysiological recordings, we investigated in hippocampal networks the impact of autoantibodies against glutamate NMDA receptor (NMDAR-Ab) on several aspects of the glutamate synapse.ResultsWe ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor. Functionally, only patients’ NMDAR-Ab prevent long-term potentiation at glutamatergic synapses while leaving NMDAR-mediated calcium influx intact. Furthermore, we unveil that NMDAR-Ab from first episode psychotic patients produced similar effects.DiscussionBy taking advantage of the single molecule imaging and complementary ensemble approaches, we unveil that NMDAR-Ab from psychotic patients (schizophrenic and first episode) profoundly alter NMDAR synaptic transmission and NMDAR-dependent synaptic functions, supporting a pathogenically relevant role.

Positive Selection on a High-Sensitivity Allele of the Human Bitter-Taste Receptor TAS2R16

During periods of human expansion into new environments, recognition of bitter natural toxins through taste may have conferred an important selective advantage. The G protein-coupled receptor encoded by TAS2R16 mediates response to salicin, amygdalin, and many bitter beta-glucopyranosides. beta-glucopyranosides are ubiquitous in nature, with many having a highly toxic cyanogenic activity. We examined evidence for natural selection on the human receptor TAS2R16 by sequencing the entire coding region, as well as part of the 5' and 3' UTRs, in 997 individuals from 60 human populations. We detected signatures of positive selection, indicated by an excess of evolutionarily derived alleles at the nonsynonymous site K172N and two linked sites and significant values of Fay and Wu's H statistics in 19 populations. The estimated age range for the common ancestor of the derived N172 variant is 78,700-791,000 years, placing it in the Middle Pleistocene and before the expansion of early humans out of Africa. Using calcium imaging in cells expressing different receptor variants, we showed that N172 is associated with an increased sensitivity to salicin, arbutin, and five different cyanogenic glycosides. We have detected a clear signal of positive selection at the bitter-taste receptor gene TAS2R16. We speculate that the increased sensitivity that is shown toward harmful cyanogenic glycosides and conferred by the N172 allele may have driven the signal of selection at an early stage of human evolution.