A Targetable Fluorescent Sensor Reveals That Copper-Deficient SCO1 and SCO2 Patient Cells Prioritize Mitochondrial Copper Homeostasis

Abstract

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 metallation 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 wildtype 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.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis

“…Mito-CS1 was synthesized as described (29). Reactivity with copper was measured by titrating the freshly diluted probe with copper.…”

Reaction-based fluorescent sensor for investigating mobile Zn 2+ in mitochondria of healthy versus cancerous prostate cells

“…In targeting mitochondria, we relied on an aminoethyl derivative of triphenylphosphine to generate the lipophilic TPP cation (31). TPP delivers payloads, including fluorescent sensors (21)(22)(23)(24)(25)(26)(27), to the mitochondrial matrix by exploiting the negative potential maintained by actively respiring mitochondria (32). The free amino group on the aminoethyl TPP derivative provided a convenient synthetic handle for attachment of 6-CO 2 H ZP1 (33).…”

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

“…This complex has been defined as the copper ligand (CuL), and its existence and localization have since been confirmed by x-ray fluorescence imaging and copper chelation studies (12,13). Copper-dependent human SOD1 localized to this mitochondrial compartment is able to rescue a range of phenotypic defects associated with SOD2 deletion, demonstrating the accessibility of this pool (11).…”