Organelle Calcium Function in Endothelial Cells

As in virtually every cell type, Ca2+ operates as a crucial messenger for numerous pivotal functions in endothelial cells. Such exciting variability of Ca2+ is even more impressive considering the precision of the regulation of Ca2+- sensitive mechanisms. But how can a molecule as simple as Ca2+ be the mediator of such a complex and precise machinery and how sensitive is it to pathological conditions? In our previous grant, we demonstrated that in endothelial cells the paradox of multiple but still highly specific actions of Ca2+ is solved, at least in part, by locally restricted Ca2+ gradients in the subplasmalemmal cytosol. There, domains of the endoplasmic reticulum achieve spatial Ca2+ elevations of >6.5 M upon cell stimulation and thus, stimulate Ca2+-activated ion channels and enzymes. In contrast, in the vicinity of superficial mitochondria, Ca2+ remains basal (~0.2 M), which is essential to maintain Ca2+-inihibitable capacitative Ca2+ entry. Preliminary data point to an existence of inter-organelle Ca2+ crosstalk as a key phenomenon in Ca2+ homeostasis. Moreover, organelle structure is matter of continuous changes due to tubular movements, fusion and fission. The mechanisms, regulation and impact of interplay, shape and dynamics of organelles, and the orchestration of ion movements across organelles and the plasma membrane are unclear and will be explored in this project. To achieve a detailed evaluation of spatial and organelle Ca2+ signaling and organelle dynamics for selective Ca2+- regulation of cell functions, new molecular tools based on e.g. fluorescence resonance energy transfer will be designed and utilized in experiments in which electrophysiology and array laser scanning confocal microscopy is performed simultaneously. Despite strong evidence that a distorted Ca2+ homeostasis accounts, at least in part, for endothelial dysfunction promoting vascular complications in e.g. diabetes mellitus, a detailed analysis of molecular and local aspects of altered Ca2+ signaling is missing. Based on recent concepts of the regulation of spatial Ca2+ signaling and the improvements in monitoring spatial Ca2+ signaling, this project is further aimed to assess the mechanisms and consequences of alterations in spatial and organelle Ca2+ signaling/dynamics in endothelial cells during e.g. hyperglycemic conditions.

As in virtually every cell type, Ca2+ operates as a crucial messenger for numerous pivotal functions in endothelial cells. Such exciting variability of Ca2+ is even more impressive considering the precision of the regulation of Ca2+- sensitive mechanisms. But how can a molecule as simple as Ca2+ be the mediator of such a complex and precise machinery and how sensitive is it to pathological conditions? In our previous grant, we demonstrated that in endothelial cells the paradox of multiple but still highly specific actions of Ca2+ is solved, at least in part, by locally restricted Ca2+ gradients in the subplasmalemmal cytosol. There, domains of the endoplasmic reticulum achieve spatial Ca2+ elevations of >6.5 M upon cell stimulation and thus, stimulate Ca2+-activated ion channels and enzymes. In contrast, in the vicinity of superficial mitochondria, Ca2+ remains basal (~0.2 M), which is essential to maintain Ca2+-inihibitable capacitative Ca2+ entry. Preliminary data point to an existence of inter- organelle Ca2+ crosstalk as a key phenomenon in Ca2+ homeostasis. Moreover, organelle structure is matter of continuous changes due to tubular movements, fusion and fission. The mechanisms, regulation and impact of interplay, shape and dynamics of organelles, and the orchestration of ion movements across organelles and the plasma membrane are unclear and will be explored in this project. To achieve a detailed evaluation of spatial and organelle Ca2+ signaling and organelle dynamics for selective Ca2+-regulation of cell functions, new molecular tools based on e.g. fluorescence resonance energy transfer will be designed and utilized in experiments in which electrophysiology and array laser scanning confocal microscopy is performed simultaneously. Despite strong evidence that a distorted Ca2+ homeostasis accounts, at least in part, for endothelial dysfunction promoting vascular complications in e.g. diabetes mellitus, a detailed analysis of molecular and local aspects of altered Ca2+ signaling is missing. Based on recent concepts of the regulation of spatial Ca2+ signaling and the improvements in monitoring spatial Ca2+ signaling, this project is further aimed to assess the mechanisms and consequences of alterations in spatial and organelle Ca2+ signaling/dynamics in endothelial cells during e.g. hyperglycemic conditions.