Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) to in vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80 -90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.copper chelation ͉ human microvascular endothelial cells ͉ infiltrating ductal breast carcinoma E ndogenous metals, such as Cu, Fe, and Zn, are subject to complex regulation in cellular systems. They are required as cofactors or regulators of numerous proteins (1) and yet, if present in overabundance, are toxic and expose the cellular environment to adventitious redox activity (2). This delicate balance is thought to be achieved by sequestration of these metals within their target proteins, metallochaperone systems, or distinct subcellular compartments (3). Although many proteins that handle transition metals within cells have been identified (4), our knowledge as to how metal content is dynamically regulated in eukaryotic cells is still limited. To what extent does regulation of the metal ion content of individual metalloproteins, mediated by protein-protein interactions, serve as an additional level of regulation of cellular metalloprotein activity? Could such regulation result in polarization of transition metal distribution throughout a cell during a biological process? To begin to explore such questions, we examined a cellular system whose biology is acutely sensitive to modulation by metals.

Summary Carotenoids are vital phytonutrients widely recognised for their health benefits. Therefore, it is vital to thoroughly investigate the metabolic regulatory network underlying carotenoid biosynthesis and accumulation to open new leads towards improving their contents in vegetables and crops. The outcome of our study defines SlWRKY35 as a positive regulator of carotenoid biosynthesis in tomato. SlWRKY35 can directly activate the expression of the 1‐deoxy‐d‐xylulose 5‐phosphate synthase (SlDXS1) gene to reprogramme metabolism towards the 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway, leading to enhanced carotenoid accumulation. We also show that the master regulator SlRIN directly regulates the expression of SlWRKY35 during tomato fruit ripening. Compared with the SlLCYE overexpression lines, coexpression of SlWRKY35 and SlLCYE can further enhance lutein production in transgenic tomato fruit, indicating that SlWRKY35 represents a potential target towards designing innovative metabolic engineering strategies for carotenoid derivatives. In addition to providing new insights into the metabolic regulatory network associated with tomato fruit ripening, our data define a new tool for improving fruit content in specific carotenoid compounds.

We developed a two-photon fluorescence probe for the dynamic and reversible detection of O2˙– levels in the Golgi apparatus.

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