The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signalling

The primary cilium is an organelle present in most adult mammalian cells and is thought of as an antenna for detection of a variety of signals. Here we use intact mouse pancreatic islets of Langerhans to investigate signalling properties of the primary cilium in β-cells. Using cilia-targeted Ca2+ indicators we find that the resting Ca2+ concentration in the cilium is lower than that of the cytosol, and we uncover a Ca2+ extrusion mechanism in the cilium that effectively insulates the cilium from changes in cytosolic Ca2+. Stimuli that give rise to pronounced cytosolic Ca2+ concentration increases, such as glucose- and depolarization-induced Ca2+ influx, and mobilization of Ca2+ from the ER, was accompanied by minor increases in cilia Ca2+ concentrations that were spatially restricted to a small compartment at the base. Conversely, we observe pronounced Ca2+ concentration changes in the primary cilia of islet β-cells that do not propagate into the cytosol and show that paracrine GABA signalling via cilia-localized GABA- B1-receptors is responsible for this Ca2+ signalling. Finally, we demonstrate that the cilia response to GABA involves ligand-dependent transport of GABA-B1 receptors into the cilium.

Keratin 7 Is a Constituent of the Keratin Network in Mouse Pancreatic Islets and Is Upregulated in Experimental Diabetes

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 β-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of β-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ β-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse β-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.

AK247, AK249, AK250, AK280 and AK281 antibodies label mouse glucagon-secreting alpha cells by immunohistochemistry

The recombinant antibodies AK247, AK249, AK250, AK280 and AK281 detect by immunohistochemistry the glucagon-secreting alpha cells in mouse pancreatic islets.

Harnessing reaction-based probes to preferentially target pancreatic β-cells and β-like cells

Highly sensitive approaches to target insulin-expressing cells would allow more effective imaging, sorting, and analysis of pancreatic β-cells. Here, we introduce the use of a reaction-based probe, diacetylated Zinpyr1 (DA-ZP1), to image pancreatic β-cells and β-like cells derived from human pluripotent stem cells. We harness the high intracellular zinc concentration of β-cells to induce a fluorescence signal in cells after administration of DA-ZP1. Given its specificity and rapid uptake by cells, we used DA-ZP1 to purify live stem cell-derived β-like cells as confirmed by immunostaining analysis. We tested the ability of DA-ZP1 to image transplanted human islet grafts and endogenous mouse pancreatic islets in vivo after its systemic administration into mice. Thus, DA-ZP1 enables purification of insulin-secreting β-like cells for downstream applications, such as functional studies, gene-expression, and cell–cell interaction analyses and can be used to label engrafted human islets and endogenous mouse islets in vivo.

Isotropic 3D electron microscopy reference library of whole cells and tissues

SummaryUnderstanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structure with nanometer resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations inasmuch as they only visualize a single slice or a relatively small volume of the cell, respectively. Here, we overcome these limitations by imaging whole cells and tissues with enhanced Focus Ion Beam Scanning Electron Microscopy (FIB-SEM) in high resolution with month-long acquisition duration. We use this approach to generate reference 3D image datasets at 4-nm isotropic voxels for ten different examples, including cultured cells (cancer, macrophages, and T-cells) as well as tissues (mouse pancreatic islets and the Drosophila fan-shaped body). We open access to all datasets in OpenOrganelle, an interactive web platform that allows accessing both the original 3D EM data, and subsequent organelle segmentation. Together, these data will serve as a reference library to explore comprehensive quantification of whole cells and their constituents, thus addressing questions related to cell identities, cell morphologies, cell-cell interactions, as well as intracellular organelle organization and structure.