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Intact mitochondrial Ca uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

Mitochondrial Ca uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO) production. However, it is not entirely clear if the organelles support or counteract NO biosynthesis by taking up Ca. The objective of this study was to verify whether or not mitochondrial Ca uptake influences Ca-triggered NO generation by endothelial NO synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO probes, the geNOps, and Ca sensors to monitor single cell NO and Ca dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP)-generating agonist. Mitochondrial Ca uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca-triggered NO increase independently of global cytosolic Ca signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca sequestration and Ca-induced NO signals. The positive correlation between mitochondrial Ca elevation and NO production was independent of eNOS phosphorylation at serine. Our findings emphasize that manipulating mitochondrial Ca uptake may represent a novel strategy to control eNOS-mediated NO production.

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Individually addressable multi-chamber electroporation platform with dielectrophoresis and alternating-current-electro-osmosis assisted cell positioning.

A multi-functional microfluidic platform was fabricated to demonstrate the feasibility of on-chip electroporation integrated with dielectrophoresis (DEP) and alternating-current-electro-osmosis (ACEO) assisted cell/particle manipulation. A spatial gradient of electroporation parameters was generated within a microchamber array and validated using normal human dermal fibroblast (NHDF) cells and red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs) with various fluorescent indicators. The edge of the bottom electrode, coinciding with the microchamber entrance, may act as an on-demand gate, functioning under either positive or negative DEP. In addition, at sufficiently low activation frequencies, ACEO vortices can complement the DEP to contribute to a rapid trapping/alignment of particles. As such, results clearly indicate that the microfluidic platform has the potential to achieve high-throughput screening for electroporation with spatial control and uniformity, assisted by DEP and ACEO manipulation/trapping of particles/cells into individual microchambers.

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