Sheel C. Dodani

The transition metal copper (Cu) is essential for all living organisms but is toxic when present in excess. To identify Cu deficiency responses comprehensively, we conducted genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of a mutant defective in the gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), which acts as a transcriptional regulator of Cu deficiency responses. In response to Cu deficiency, FERRIC REDUCTASE OXIDASE5 (FRO5) and FRO4 transcript levels increased strongly, in an SPL7-dependent manner. Biochemical assays and confocal imaging of a Cu-specific fluorophore showed that high-affinity root Cu uptake requires prior FRO5/FRO4-dependent Cu(II)-specific reduction to Cu(I) and SPL7 function. Plant iron (Fe) deficiency markers were activated in Cu-deficient media, in which reduced growth of the spl7 mutant was partially rescued by Fe supplementation. Cultivation in Cu-deficient media caused a defect in root-to-shoot Fe translocation, which was exacerbated in spl7 and associated with a lack of ferroxidase activity. This is consistent with a possible role for a multicopper oxidase in Arabidopsis Fe homeostasis, as previously described in yeast, humans, and green algae. These insights into root Cu uptake and the interaction between Cu and Fe homeostasis will advance plant nutrition, crop breeding, and biogeochemical research.

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu(+)-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu(+) in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu(+) pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wild-type controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.

We present the synthesis and properties of Nickelsensor-1 (NS1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively responding to Ni(2+) in aqueous solution and in living cells. NS1 combines a BODIPY chromophore and a mixed N/O/S receptor to provide good selectivity for Ni(2+) over a range of biologically abundant metal ions in aqueous solution. In addition to these characteristics, confocal microscopy experiments further show that NS1 can be delivered into living cells and report changes in intracellular Ni(2+) levels in a respiratory cell model.

Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.