Cytosolic Ca2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2+/H+ antiporter that achieved mitochondrial Ca2+ sequestration at small Ca2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca2+ but strongly diminished the transfer of entering Ca2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca2+ uptake at low Ca2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca2+ uptake pathways in intact endothelial cells.

Highlights► Mitochondrial Ca2+ sequestration was tested by various techniques. ► Kinetics and Ca2+ sensitivity of mitochondrial Ca2+ uptake depends on the techniques chosen. ► By electrophysiology, 2 and 3 distinct Ca2+ inward currents were measured in HeLa and endothelial cell mitoplasts. ► Mitoplast Ca2+ inward currents differ in frequency of appearance and conductance ranging from 7.6 to 74.3 pS. ► Mitochondrial Ca2+ uptake routes/modes exists and might be cell specific.

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca2+ uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca2+ currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca2+ as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca2+ in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K+-channel inhibitors, and Cl− channel blockers disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca2+ currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca2+ uniporter.

BackgroundImproving our understanding of cancer and other complex diseases requires integrating diverse data sets and algorithms. Intertwining in vivo and in vitro data and in silico models are paramount to overcome intrinsic difficulties given by data complexity. Importantly, this approach also helps to uncover underlying molecular mechanisms. Over the years, research has introduced multiple biochemical and computational methods to study the disease, many of which require animal experiments. However, modeling systems and the comparison of cellular processes in both eukaryotes and prokaryotes help to understand specific aspects of uncontrolled cell growth, eventually leading to improved planning of future experiments. According to the principles for humane techniques milestones in alternative animal testing involve in vitro methods such as cell-based models and microfluidic chips, as well as clinical tests of microdosing and imaging. Up-to-date, the range of alternative methods has expanded towards computational approaches, based on the use of information from past in vitro and in vivo experiments. In fact, in silico techniques are often underrated but can be vital to understanding fundamental processes in cancer. They can rival accuracy of biological assays, and they can provide essential focus and direction to reduce experimental cost.Main bodyWe give an overview on in vivo, in vitro and in silico methods used in cancer research. Common models as cell-lines, xenografts, or genetically modified rodents reflect relevant pathological processes to a different degree, but can not replicate the full spectrum of human disease. There is an increasing importance of computational biology, advancing from the task of assisting biological analysis with network biology approaches as the basis for understanding a cell’s functional organization up to model building for predictive systems.ConclusionUnderlining and extending the in silico approach with respect to the 3Rs for replacement, reduction and refinement will lead cancer research towards efficient and effective precision medicine. Therefore, we suggest refined translational models and testing methods based on integrative analyses and the incorporation of computational biology within cancer research.

Background: Mitochondria may utilize different proteins to decode high and low cytosolic Ca2+.Results: Inhibition of SERCA shifts mitochondrial Ca2+ uptake from being UCP3-dependent to Letm1-dependent.Conclusion: Depending on the mode of intracellular Ca2+ release, two different mitochondrial Ca2+ uptake pathways are engaged.Significance: The dissection of two molecularly distinct mitochondrial Ca2+ uptake routes depending on SERCA activity points to the complexity of the mitochondrial Ca2+ uptake machinery.