CONTENTS 1. Introduction 4564 2. General Features of Fluorescent Sensors for Metal Ions 4566 2.1. Photophysical Properties of Fluorophores 4566 2.2. Mechanisms of Altering a Fluorescence Signal 4566 2.3. Classes of Sensors for Live-Cell Imaging 4568 2.3.1. Molecular Probes 4568 2.3.2. Genetically Encoded Probes 4568 2.3.3. Hybrid Probes 4569 3. Important Considerations for Introduction of Sensors 4569 3.1. Factors Affecting the Intracellular Concentration of Sensors 4569 3.2. Buffering 4570 3.3. Localization 4570 3.3.1. Factors Governing Localization of Molecular Probes 4571 3.3.2. Genetic Targeting of Probes 4572 4. A Brief History of Visualizing Cellular Metal Ion Distribution with Probes 4572 5. Probes for Zinc 4576 5.1. Zinc Homeostasis 4576 5.2. Small-Molecule Probes for Zn 2+ 4576 5.2.1. Intensity-Based Probes 4576 5.2.2. Ratiometric Probes for Zn 2+ 4580 5.3. Genetically Encoded Sensors for Zn 2+ 4581 5.4. Hybrid Probes for Zn 2+ 4583 6. Probes for Copper 4584 7. Probes for Iron 4587 7.1. Iron Homeostasis 4587 7.2. Early Probes for Labile Iron 4588 7.3. Probes for Fe 2+ 4588 7.4. Probes for Fe 3+ 4589 8. Available Fluorescent Probes for Other Biological Metals 4591 8.1. Manganese (Mn 2+ ) 4591 8.2. Nickel (Ni 2+ ) 4592 8.3. Cobalt (Co 2+ ) 4592 9. Probes for Toxic Metals 4592 9.1. Lead (Pb 2+ ) 4592 9.2. Cadmium (Cd 2+ ) 4594 9.3. Mercury (Hg 2+ ) 4594 10. Outlook 4596 Author Information 4596 Corresponding Author 4596 Notes 4596 Biographies 4597 References 4597

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond, and those that do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop novel drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs less activity towards metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc-chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a de-ubiquinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin-dependent sorting of cell-surface receptors, and Brcc36, a K63-specific deubiquitnase of BRCC36-containing isopeptidase complex (BRISC) and BRCA1-BRCA2-containing complex (BRCC). We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically-encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic Reprints and permissions information is available at www.nature.com/reprints.