Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy

Abstract

Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of ⌬G ‡ ‫؍‬ 44 kJ⅐mol ؊1 and k obs ϳ 10 5 s ؊1 at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.photoinduced electron transfer ͉ metal exchange kinetics ͉ dynamic NMR ͉ microprobe x-ray absorption near-edge spectroscopy

X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis

“…Copper was consistently centrally localized to an area at the periphery of the nucleus (Fig. 1, Flat), consistent with the copper topography observed in other cell types (data not shown) and consistent with reports of some colocalization of copper with Golgi and mitochondrial organelles (21). When cells are stimulated with VEGF and bFGF and plated onto the basement membrane surface, copper translocates outwards toward the periphery of the cell within 30-60 min (Fig.…”

“…Most of the initial biological applications of XFM analysis to date have focused on the distribution of nonendogenous metals within the cellular space, such as the localization of TiO 2 -labeled oligonucleotides (17), the examination of platinum oxidation states (18), and bacterial uptake of chromium (19). However, more recent work has begun to demonstrate the utility of this technology for the quantitation and localization of endogenous metals, such as in studies on the phagosome environment in pathogen-infected macrophages (20), on the copper content and topography of fibroblasts (21), and on the relocalization of zinc during early stages of macrophage differentiation (22). We have used XFM to explore the relationship between copper and angiogenesis both in vitro and in vivo and have discovered that massive relocalization of cellular copper stores appears to be a requirement for proper angiogenic network formation.…”

Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

“…However, the presence of an intracellular copper pool localized in the Golgi apparatus and mitochondria (36), may allow for intracellular labeling without addition of exogenous Cu(I). Along with the development of new cell-permeable probes, labeling may be achieved in living cells without toxicity.…”

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).…”