Ratiometric imaging of flux dynamics of cobalt with an optical sensor

Highlights

• A genetically encoded nanosensor is designed for real-time measurement of cobalt.

• The senor was successfully expressed and characterized using the FRET method.

• The pH-stable sensor binds to cobalt with high sensitivity and selectivity.

• The developed sensor non-destructively detects the cellular dynamics of cobalt flux.

• The sensor applies for prokaryotic and eukaryotic systems at the single-cell level.

Abstract

Cobalt (Co²⁺) is a vital micronutrient needed for growth and development by all life types. In humans, cobalt is cytotoxic and genotoxic associated with various pathological and physiological conditions such as allergic dermatitis, pneumonia, interstitial fibrosis, alveolitis, myocardiopathy, rhinitis and lung cancer. In this study, we present a genetically encoded fluorescence resonance energy transfer (FRET)-based Cobalt Optical Sensor (CobOS) to determine this metal ion’s trafficking scheme consisting of uptake/efflux mechanisms and measuring the real time free-ion concentration and distribution within living cells. CobOS comprise of a cobalt-sensing domain CbiK^P from Desulfovibrio vulgaris Hildenborough fused with the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus at N- and C-terminus respectively. When studied for its specificity and selectivity, CobOS provides maximum ratiometric readout for cobalt ions, thereby establishing its effectiveness as a FRET cobalt sensor. The sensor is resistant to pH changes and remains unaffected by adding other metal ions that are biologically significant. CobOS-86n, the most efficient sensor variant, binds cobalt with an affinity (K_d) of 0.86 × 10⁻⁶ M covering cobalt concentrations of 50 nM to 200 μM and can be of practical interest for real time measurement of in vivo cobalt levels in Escherichia coli (E. coli), yeast and mammalian cells non-invasively using widefield confocal fluorescent microscopy.

Introduction

In many molecular processes, transition metal ions function as important cofactors catalysing various enzymatic biochemical reactions. Trace metal ions play critical biological roles within the cells, not only preserving molecular integrity, but also identifying the structural and signalling mechanisms engaged in different cellular processes [1]. Cells acquire metal ions either by passive absorption or actively by creating an energy-dependent electrical potential through ATPases [2]. Cobalt ions are usually present in cells at comparatively reduced levels, performing significant structural and functional roles to control homeostasis within the cell, thereby avoiding toxicity that happens when their concentration exceeds standard physiological threshold [3]. Cobalt ions are needed in the form of vitamin, cyanocobalamin (Vitamin B₁₂) that is not readily absorbed in the body [4,5]. This metalloenzyme needs cobalt, placed in the centre of the corrin ring of the tetra-pyrrole. Cobalt enters the body in a number of ways: first, with food; second, with the respiratory system; third, with the skin; and finally, as a biomaterial element [5]. It was also observed that no adverse responses in humans were associated with 300 μg/L and less blood cobalt concentrations [6] suggesting that cobalt concentration of 5.09 μM or less is considered non-toxic to the human cells. Cobalt plays an indispensable part in the formation of red blood cells, the metabolism of fat and carbohydrates, the catalysis of enzymatic reactions engaged in nucleic acid and neurotransmitter synthesis, and the synthesis of proteins engaged in the formation of myelin sheath. It is also known to play a role in the transport of blood glucose into the body cells, aids in iron absorption, neuromuscular and digestive disorders and radioactive cobalt for the treatment of certain cancers, sterilization of medical equipment and implanted medical devices such as knee and hip replacements [4,5,7]. Insufficient cobalt levels result in inadequate vitamin B₁₂ synthesis, leading to pernicious anaemia, nerve and thyroid diseases, adverse effects on bone formation and resorption, enhanced fetal, developmental and cellular abnormalities, scaly skin and atrophy [2,5]. Excess of this metal ion, however, indicates negative health conditions that cause respiratory disorders, lung fibrosis and asthma, polycythaemia, heart failure and cardiomyopathy, enhanced bone marrow activity [1]. Cobalt ions form reactive species in vivo and inhibit processes for DNA repair, resulting in genotoxicity and carcinogenicity [8].

Understanding the metabolic dynamics of cobalt ions becomes a need to elucidate the different cellular, molecular, biochemical elements of cobalt physiology. Prokaryotes have effective uptake and efflux systems in the form of transporters to balance the concentration of cobalt ions in the cell [1]. Some ABC systems, consisting of three (CbiMQO) or four (CbiMNQO) components and encoded within clusters of the prokaryotic coenzyme B₁₂ biosynthesis gene are thought to be involved in cobalt uptake [9]. NhlF from Rhodococcus rhodochrous J1 and HupE/UreJ homologs in cyanobacteria prefers transport of cobalt. The transporter genes in these organisms are regulated by a coenzyme B₁₂-dependent riboswitch element and are therefore linked to cobalt metabolism. It has been stated that the COT2 gene of Saccharomyces cerevisiae (S. cerevisiae) is not responsible for cobalt transport but may play a more general role in yeast physiology, which indirectly regulates the permeability of the membrane to cobalt ions or the driving force for the uptake in yeast. On the other hand, both NhlF and COT1 contain a histidine residue which is a possible metal-binding amino acid that indicate the functional role for the cobalt-specific recognition [10]. Classical methods like nuclear magnetic resonance (NMR), gas chromatography–mass spectroscopy (GCMS), liquid chromatography–mass spectroscopy (LCMS), capillary electrophoresis-mass spectroscopy (CEMS), ultra-performance liquid chromatography–mass spectroscopy (UPLC-MS), atomic absorption spectroscopy (AAS), high performance liquid chromatography–mass spectroscopy (HPLC-MS), atomic pressure ionization (API), electron spray ionisation (ESI) are widely used to monitor Co²⁺ in real time [[11], [12], [13]]. Obtaining metabolic profile needs tissue fractionation and comprehensive preparation of samples that degrades the natural healthy state of the cells [14,15]. These methods are invasive, less selective and less precise, have restricted resolution and demonstrate low sensitivity. Chemosensors for Co²⁺ detection usually employs two types of response mechanisms-colorimetric and fluorometric. Colorimetric methods are convenient, having attracted considerable attention can easily monitor target ions with the naked eye [16]. One such sensor uses functionalized silver nanoparticles (DDTC-Ag NPs) of dopamine dithiocarbamate as the sensing material and is based on unique surface plasmon resonance properties. The presence of Co²⁺ induces aggregation of Ag NPs by coordinating covalent bonds between the DDTC catechol groups on the surface of Ag NPs and cobalt ions, resulting in a change of color from bright yellow to red [17]. Furthermore, fluorescence probes have also emerged to be promising candidates for the detection of Co²⁺ due to their high specificity against target compounds, great simplicity and high-speed analysis during the experiment phase. A Co²⁺ chemosensor based on 1,8-naphthalimide appending thiourea has been designed and synthesised successfully. The fluorescent chemosensor (L), using turn-on fluorescence enhancement with visual colorimetric response, detected the presence of Co²⁺ [16]. In another such sensor, fluorescent _L-Tyr capped silver nanoparticles (AgNPs) have been used for Co²⁺ trace-level detection using the chelation-assisted fluorescence enhancement method for detection [18].

Molecular live-cell imaging is gaining tremendous popularity extending the horizon from visualization to exploration of molecular structures and biological processes [19,20]. The advent of fluorescence technology along with novel fluorescent probes makes live cell imaging a reliable and efficient way to study cobalt physiology [21]. The detection approaches however come with its own disadvantages. Some of these probes suffer from toxicity, low bio-compatibility and complicated synthetic processes which may impede their practical use in related fields [16]. Organic dyes are susceptible to metabolic degradation, have minimal photostability and show reduced quantum yield near-infrared wavelengths. Their use in the dual emission detectors results in bleed-through or spectral crosstalk. Because of their relatively narrow absorption/emission bands and small Stokes shifts, organic dyes experience a spectrum of 'red tail' emissions, resulting in signal leakage [22]. On the other hand, quantum dots (QDs) are cytotoxic nanocolloids, and their large absorption spectra further limit their use as FRET acceptors in fluorescence quantification studies. The QD-protein fusion sensors make them less effective and difficult to induce in living cells because of their potentially large size and random switching between emitting and non-emitting states [[22], [23], [24]]. Consequently, the search for a novel and concise probe is still in urgent demand for the further and efficient detection of Co²⁺. The replacement of toxic dyes with spectral variants of green fluorescent protein (GFP) is therefore an important approach that we have adopted to limit its drawbacks.

Here, we report the construction of genetically encoded cobalt nanosensor based on FRET that enables keeping a track on pathways involving this metal ion. ECFP and yellow fluorescent protein variant Venus is the FRET pair used to sandwich a cobalt-binding periplasmic gene, cbik^p for nanosensor development. A sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, has a cobaltochelatase activity that catalyses the incorporation of Co²⁺ into sirohydrochlorine. The cobalt-binding domain undergoes a conformational change upon binding of Co²⁺ altering the distance between the two fluorophores giving a signal in the form of fluorescence. This sensor is genetically encoded and potentially non-invasive, establishing its effectiveness and can be used cellular systems for detection of Co²⁺ with high spatio-temporal resolution. This nanosensor can be used to detect and measure intracellular levels of cobalt ions in a single cell and to monitor the flow of this metal ion in bacterial cells and eukaryotes such as yeast and animal cells.