Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells

Abstract

Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer

“…Depending on its kinetic accessibility, cellular Cu can be divided into a canonical, static pool (i.e., Cu bound to enzymes such as cytochrome c oxidase or SOD1) and an exchangeable, labile pool (i.e., Cu bound to chaperones such as CCS) 24 . The latter form of Cu is more bioavailable and can participate in dynamic cell signaling pathways 25 .…”

Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae

“…Fahrni and coworkers used chelators to reveal a small pool of tightly bound copper coordinated to thiols. 20 The concentration of this pool increases transiently after cells are exposed to copper salts, especially in mitochondria and Golgi. 21 Also interesting is that the phenotypes of cells lacking various copper chaperones (e.g.…”

l-Cysteine-mediated modulation of copper trafficking in prostate cancer cells: an in vitro and in vivo investigation with 64Cu and 64Cu-PET

“…The fast onset of the effect precludes the involvement of protein synthesis in the process. Since the intracellular copper ions are bound with attomolar affinities, 65 it is unlikely that copper-Lcysteine complexes with their much lower stability constants 66 would be able to account for intracellular buffering of copper. In another line of explanation, we suggest that L-cysteine exerted an indirect effect on 64 Cu trafficking, which increased 64 Cu flux to the retention sites.…”