Coupling of calcium (Ca²⁺) release by ryanodine receptor (RyR) 3 to its extrusion by sodium/calcium exchanger (NCX) mediates endoplasmic reticulum (ER) Ca²⁺ discharge from vascular endothelial cells

The endoplasmic reticulum (ER) is an organelle with multiple functions serving to maintain cellular health, the most important of which include its role as the cell’s largest store of the signaling ion calcium (Ca²⁺). The regulation of this ion’s movement into and out of the ER, as well as the cell itself, is therefore of the utmost importance when considering the overarching goal of each cellular body to remain functional. The study outlined in the current thesis discusses hypotheses for the manner in which Ca²⁺’ is released in a dramatic dysregulation event during ER stress, a cellular condition that often results in cell death and which has been implicated in multiple severe health conditions. This pathway was chosen to be studied in endothelial cells (ECs), those comprising the intimal (inner-most) layer of blood vessels. These cells are in direct contact with blood flowing through these tributaries of the vasculature, and their vast array of products controls constriction and many other properties of the blood vessel itself. Dysfunction of these cells results in the loss of their ability to exert protective effects on the affected blood vessel. Importantly, this type of damage to ECs can occur as a result of Ca²⁺ dysregulation at the level of the ER, such as the dramatic ER Ca²⁺ drop being studied here. It is therefore the aim of the current thesis to present evidence for the mechanism whereby dramatic changes in Ca²⁺ levels within the ER lead to problems cellular function, with the ultimate goal of therapy development to prevent affected cells from becoming dysfunctional as a result of such ion dysregulation. Investigation into the way that cytoplasmic and luminal ER Ca²⁺ levels are affected by a range of agonists as well as relevant channel and pump blockers has culminated in a clearer understanding of the likely pathway for this Ca²⁺ movement early in the ER stress response, which may in future be manipulated at the point of ER stress induction to potentially deter subsequent cell failure, and ultimately, vascular disease.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Tracking intra- and inter-organelle signaling of mitochondria

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca2+ and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine‐tuned by intra‐ and interorganelle H+, K+, Na+, and Ca2+ signaling. In this review, we provide an up‐to‐date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting‐edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.ISSN:1742-464XISSN:1742-465

Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors

Mitochondrial Ca2+ uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca2+ kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca2+ biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca2+-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca2+ levels inside the CL but not the IMS in response to IP3-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca2+ through the OMM while the cytosolic Ca2+ signal remained unchanged. The novel sub-mitochondrially targeted Ca2+ biosensors proved to be suitable for Ca2+ imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca2+ signaling under (patho)physiological conditions.ISSN:1662-510

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose

BACKGROUND/AIMS: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes