Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K+, alkaline earth metals including Mg2+ and Ca2+, and transition metals including Cu+/Cu2+ and Zn2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe2+/Fe3+, Mn2+ and Na+.

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Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds

PLANT PHYSIOLOGY ◽

10.1104/pp.106.082586 ◽

2006 ◽

Vol 141 (4) ◽

pp. 1446-1458 ◽

Cited By ~ 200

Author(s):

Brian M. Waters ◽

Heng-Hsuan Chu ◽

Raymond J. DiDonato ◽

Louis A. Roberts ◽

Robynn B. Eisley ◽

...

Keyword(s):

Metal Ions ◽

Metal Ion ◽

Ion Homeostasis ◽

Metal Ion Homeostasis ◽

Yellow Stripe

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The role of metal ions in the virulence and viability of bacterial pathogens

Biochemical Society Transactions ◽

10.1042/bst20180275 ◽

2019 ◽

Vol 47 (1) ◽

pp. 77-87 ◽

Cited By ~ 16

Author(s):

Stephanie L. Begg

Keyword(s):

Metal Ions ◽

Metal Ion ◽

Molecular Mechanisms ◽

Bacterial Pathogens ◽

Ion Homeostasis ◽

Transition Metal Ions ◽

Metal Ion Homeostasis ◽

And Function ◽

Iron Manganese

AbstractMetal ions fulfil a plethora of essential roles within bacterial pathogens. In addition to acting as necessary cofactors for cellular proteins, making them indispensable for both protein structure and function, they also fulfil roles in signalling and regulation of virulence. Consequently, the maintenance of cellular metal ion homeostasis is crucial for bacterial viability and pathogenicity. It is therefore unsurprising that components of the immune response target and exploit both the essentiality of metal ions and their potential toxicity toward invading bacteria. This review provides a brief overview of the transition metal ions iron, manganese, copper and zinc during infection. These essential metal ions are discussed in the context of host modulation of bioavailability, bacterial acquisition and efflux, metal-regulated virulence factor expression and the molecular mechanisms that contribute to loss of viability and/or virulence during host-imposed metal stress.

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Metal ions, Alzheimer's disease and chelation therapy

Acta Pharmaceutica ◽

10.2478/v10007-011-0006-6 ◽

2011 ◽

Vol 61 (1) ◽

pp. 1-14 ◽

Cited By ~ 110

Author(s):

Ana Budimir

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Metal Ions ◽

Metal Ion ◽

Chelation Therapy ◽

Neurological Diseases ◽

Ion Homeostasis ◽

Ionic Balance ◽

Metal Ion Homeostasis ◽

Ion Imbalance

Metal ions, Alzheimer's disease and chelation therapyIn the last few years, various studies have been providing evidence that metal ions are critically involved in the pathogenesis of major neurological diseases (Alzheimer, Parkinson). Metal ion chelators have been suggested as potential therapies for diseases involving metal ion imbalance. Neurodegeneration is an excellent target for exploiting the metal chelator approach to therapeutics. In contrast to the direct chelation approach in metal ion overload disorders, in neurodegeneration the goal seems to be a better and subtle modulation of metal ion homeostasis, aimed at restoring ionic balance. Thus, moderate chelators able to coordinate deleterious metals without disturbing metal homeostasis are needed. To date, several chelating agents have been investigated for their potential to treat neurodegeneration, and a series of 8-hydroxyquinoline analogues showed the greatest potential for the treatment of neurodegenerative diseases.

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Golgi Metal Ion Homeostasis in Human Health and Diseases

Cells ◽

10.3390/cells11020289 ◽

2022 ◽

Vol 11 (2) ◽

pp. 289

Author(s):

Jie Li ◽

Yanzhuang Wang

Keyword(s):

Metal Ions ◽

Membrane Trafficking ◽

Metal Ion ◽

Secretory Pathway ◽

Ion Homeostasis ◽

Protein Glycosylation ◽

Proper Function ◽

Metal Ion Homeostasis ◽

Trafficking Pathway ◽

Membrane Organelle

The Golgi apparatus is a membrane organelle located in the center of the protein processing and trafficking pathway. It consists of sub-compartments with distinct biochemical compositions and functions. Main functions of the Golgi, including membrane trafficking, protein glycosylation, and sorting, require a well-maintained stable microenvironment in the sub-compartments of the Golgi, along with metal ion homeostasis. Metal ions, such as Ca2+, Mn2+, Zn2+, and Cu2+, are important cofactors of many Golgi resident glycosylation enzymes. The homeostasis of metal ions in the secretory pathway, which is required for proper function and stress response of the Golgi, is tightly regulated and maintained by transporters. Mutations in the transporters cause human diseases. Here we provide a review specifically focusing on the transporters that maintain Golgi metal ion homeostasis under physiological conditions and their alterations in diseases.

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Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

BMC Microbiology ◽

10.1186/s12866-021-02091-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

A. Sukumaran ◽

S. Pladwig ◽

J. Geddes-McAlister

Keyword(s):

Klebsiella Pneumoniae ◽

Metal Ion ◽

Ion Homeostasis ◽

Cellular Protein ◽

Pcr Analysis ◽

Phenotypic Analysis ◽

Metal Ion Homeostasis ◽

Capsule Production ◽

The Impact

Abstract Background Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

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The influence of metal-ion binding on the structure and surface composition of Sonic Hedgehog: a combined classical and hybrid QM/MM MD study

Physical Chemistry Chemical Physics ◽

10.1039/c6cp03960j ◽

2016 ◽

Vol 18 (32) ◽

pp. 22254-22265 ◽

Cited By ~ 7

Author(s):

Manuel Hitzenberger ◽

Thomas S. Hofer

Keyword(s):

Metal Ions ◽

Sonic Hedgehog ◽

Binding Sites ◽

Metal Ion ◽

Surface Composition ◽

Quantum Mechanical ◽

Ion Binding ◽

Metal Ion Binding ◽

Structural Impact

The interaction of metal ions with Shh binding-sites and their structural impact are assessed via classical and quantum mechanical simulations.

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Metallothionein: A Multifunctional Protein from Toxicity to Cancer

The International Journal of Biological Markers ◽

10.1177/172460080301800302 ◽

2003 ◽

Vol 18 (3) ◽

pp. 162-169 ◽

Cited By ~ 46

Author(s):

S.E. Theocharis ◽

A.P. Margeli ◽

A. Koutselinis

Keyword(s):

Cell Proliferation ◽

Metal Ion ◽

Defense Mechanisms ◽

Ion Homeostasis ◽

Low Molecular Weight ◽

Multifunctional Protein ◽

Metal Ion Homeostasis ◽

Potential Marker ◽

Proliferation And Apoptosis ◽

Carcinogenic Process

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.

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Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

Biochemical Journal ◽

10.1042/bj20061168 ◽

2006 ◽

Vol 400 (3) ◽

pp. 385-392 ◽

Cited By ~ 4

Author(s):

Erdeni Bai ◽

Federico I. Rosell ◽

Bao Lige ◽

Marcia R. Mauk ◽

Barbara Lelj-Garolla ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Metal Ions ◽

Binding Site ◽

Metal Ion ◽

Association Constants ◽

Ion Binding ◽

Ferric Uptake Regulator ◽

Metal Ion Binding ◽

Dimerization Domain ◽

High Affinity

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

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