Bright and sensitive red voltage indicators for imaging action potentials in brain slices and pancreatic islets.

Genetically encoded voltage indicators (GEVIs) allow the direct visualization of cellular membrane potential at the millisecond time scale. Among these, red-emitting GEVIs have been reported to support multichannel recordings and manipulation of cellular activities with reduced autofluorescence background. However, the limited sensitivity and dimness of existing red GEVIs have restricted their applications in neuroscience. Here, we report a pair of red-shifted opsin-based GEVIs, Cepheid1b and Cepheid1s, with improved dynamic range, brightness, and photostability. The improved dynamic range is achieved by a rational design to raise the electrochromic Förster resonance energy transfer efficiency, and the higher brightness and photostability are approached with separately engineered red fluorescent proteins. With Cepheid1 indicators, we recorded complex firings and subthreshold activities of neurons on acute brain slices and observed heterogeneity in the voltage­calcium coupling on pancreatic islets. Overall, Cepheid1 indicators provide a strong tool to investigate excitable cells in various sophisticated biological systems.

Quantitative parameters of static imaging and fast kinetics imaging in 18F-FDG total-body PET/CT for the assessment of histological feature of pulmonary lesions.

Background: To investigate the value of quantitative parameters related to static imaging and fast kinetics imaging of total-body (TB) 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in differentiating benign from malignant pulmonary lesions and squamous cell carcinoma (SCC) from adenocarcinoma (AC) and to analyze the correlation of each parameter with the Ki-67 index. Methods: A total of 108 patients with pulmonary lesions from July 2021 to May 2022 in the Henan Provincial People's Hospital, China, were consecutively recruited for TB 18F-FDG PET/CT in this prospective study. Static imaging parameters maximum standardized uptake value (SUVmax) and fast kinetics imaging parameters transport constant (K1), rate constants (k2), time delay (td), and fractional blood volume (vb) were calculated and compared. The area under the receiver operating characteristic (ROC) curve (AUC), Delong test, Logistic regression analyses, and Pearson correlation were used to assess diagnostic efficacy, find independent predictors and analyse correlations respectively. Results: Malignant lesions had higher SUVmax and K1 and lower vb than benign lesions, and SCC had higher SUVmax and K1 and lower td and vb than AC (all P<0.05). For the differentiation of benign and malignant lesions, SUVmax, K1, and vb were independent predictors, and AUC (SUVmax + K1+ vb) =0.909 (95% CI: 0.839-0.956), AUC (SUVmax) =0.883 (95% CI: 0.807-0.937), AUC (K1) =0.810 (95% CI: 0.723-0.879), and AUC (vb) =0.746 (95% CI: 0.653-0.825), where AUC (SUVmax + K1+ vb) was significantly different from AUC (K1), AUC (vb) (Z=3.006, 3.965, all P<0.05). For the differentiation of SCC and AC, SUVmax, K1, td, and vb were independent predictors, and AUC (SUVmax + K1+ td + vb) =0.946 (95% CI: 0.840-0.991), AUC (SUVmax) =0.818 (95% CI: 0.680-0.914), AUC (K1) =0.770 (95% CI: 0.626-0.879), AUC (vb) =0.737 (95% CI: 0.590-0.853), and AUC (td) =0.669 (95% CI: 0.510-0.791), where AUC (SUVmax + K1+ td + vb) was significantly different from AUC (SUVmax), AUC (K1), AUC (vb), and AUC (td) (Z=2.269, 2.821, 2.848, and 3.276, all P<0.05). SUVmax and K1 were moderately and mildly positively correlated with the Ki-67 index (r=0.541, 0.452, all P<0.05), respectively. Conclusions: Quantitative parameters of static imaging and fast kinetics imaging in 18F-FDG total-body PET/CT can be used to differentiate benign from malignant pulmonary lesions and SCC from AC and to assess Ki-67 expression.

The ß-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling.

The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting ß-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet ß-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.

The endoplasmic reticulum-plasma membrane tethering protein TMEM24 is a regulator of cellular Ca2+ homeostasis.

Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are sites of lipid exchange and Ca2+ transport, and both lipid transport proteins and Ca2+ channels specifically accumulate at these locations. In pancreatic ß-cells, both lipid and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24 (also known as C2CD2L) dynamically localizes to ER-PM contact sites and provides phosphatidylinositol, a precursor of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], to the PM. ß-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion, but the underlying mechanism is not known. We now show that TMEM24 only weakly interacts with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.

The PI(4)P phosphatase Sac2 controls insulin granule docking and release.

Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal ß cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.

Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution.

The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic ß-cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the ß-cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing ß-cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one ß-cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

Adipose tissue transplantation ameliorates lipodystrophy-associated metabolic disorders in seipin-deficient mice.

Seipin deficiency is responsible for type 2 congenital generalized lipodystrophy with severe loss of adipose tissue and can lead to hepatic steatosis, insulin resistance (IR), and dyslipidemia in humans. Adipose tissue secretes many adipokines that are central to the regulation of metabolism. In this study, we investigated whether transplantation of normal adipose tissue could ameliorate severe hepatic steatosis, IR, and dyslipidemia in lipoatrophic seipin knockout (SKO) mice. Normal adipose tissue from wild-type mice was transplanted into 6-wk-old SKO mice. At 4 mo after adipose tissue transplantation (AT), the transplanted fat survived with detectable blood vessels, and the reduced levels of plasma leptin, a major adipokine, were dramatically increased. Severe hepatic steatosis, IR, and dyslipidemia in SKO mice were ameliorated after AT. In addition, abnormal hepatic lipogenesis and ß-oxidation gene expression in SKO mice were improved after AT. Our results suggest that AT may be an effective treatment to improve lipodystrophy-associated metabolic disorders.

Feedback regulation of insulin secretion by extended synaptotagmin-1.

Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process.-Xie, B., Nguyen, P. M., Idevall-Hagren, O. Feedback regulation of insulin secretion by extended synaptotagmin-1.

Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Regulates Ca(2+)-Influx and Insulin Secretion from Pancreatic ß Cells.

Insulin secretion from pancreatic ß cells is regulated by the blood glucose concentration and occurs through Ca(2+)-triggered exocytosis. The activities of multiple ion channels in the ß cell plasma membrane are required to fine-tune insulin secretion in order to maintain normoglycemia. Phosphoinositide lipids in the plasma membrane often gate ion channels, and variations in the concentration of these lipids affect ion-channel open probability and conductance. Using light-regulated synthesis or depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), we found that this lipid positively regulated both depolarization- and glucose-triggered Ca(2+) influx in a dose-dependent manner. Small reductions of PI(4,5)P2 caused by brief illumination resulted in partial suppression of Ca(2+) influx that followed the kinetics of the lipid, whereas depletion resulted in marked inhibition of both Ca(2+) influx and insulin secretion.

Knock-down of ZBED6 in insulin-producing cells promotes N-cadherin junctions between beta-cells and neural crest stem cells in vitro.

The role of the novel transcription factor ZBED6 for the adhesion/clustering of insulin-producing mouse MIN6 and ßTC6 cells was investigated. Zbed6-silencing in the insulin producing cells resulted in increased three-dimensional cell-cell clustering and decreased adhesion to mouse laminin and human laminin 511. This was paralleled by a weaker focal adhesion kinase phosphorylation at laminin binding sites. Zbed6-silenced cells expressed less E-cadherin and more N-cadherin at cell-to-cell junctions. A strong ZBED6-binding site close to the N-cadherin gene transcription start site was observed. Three-dimensional clustering in Zbed6-silenced cells was prevented by an N-cadherin neutralizing antibody and by N-cadherin knockdown. Co-culture of neural crest stem cells (NCSCs) with Zbed6-silenced cells, but not with control cells, stimulated the outgrowth of NCSC processes. The cell-to-cell junctions between NCSCs and ßTC6 cells stained more intensely for N-cadherin when Zbed6-silenced cells were co-cultured with NCSCs. We conclude that ZBED6 decreases the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with NCSC, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival.

Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering.

The extended synaptotagmins (E-Syts) are ER proteins that act as Ca(2+)-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca(2+) regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca(2+) concentrations, we found that binding of E-Syt1 to the PI(4,5)P2-rich PM critically requires its C2C and C2E domains and that the EC50 of such binding is in the low micromolar Ca(2+) range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca(2+) via its influx from the extracellular medium, such as store-operated Ca(2+) entry in fibroblasts and membrane depolarization in ß-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca(2+).