Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are Ϸ25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.T he red fluorescent protein cloned from Discosoma coral (DsRed or drFP583) (1) holds great promise for biotechnology and cell biology as a spectrally distinct companion or substitute for the green fluorescent protein (GFP) from the Aequorea jellyfish (2). GFP and its blue, cyan, and yellow variants have found widespread use as genetically encoded indicators for tracking gene expression and protein localization and as donor͞ acceptor pairs for f luorescence resonance energy transfer (FRET). Extending the spectrum of available colors to red wavelengths would provide a distinct label for multicolor tracking of fusion proteins, and together with GFP (or a suitable variant) would provide a FRET donor͞acceptor pair that should be superior to the currently preferred cyan͞yellow pair (3). However, the evolution of DsRed from a scientific curiosity to a generally applicable and robust tool has been hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization (4). Most previous attempts to address the rate and͞or extent of maturation of DsRed (5, 6), including the commercially available DsRed2 (CLONTECH), have provided only modest improvements. However, an engineered variant of DsRed, known as T1 (see Fig. 1A), has recently become available and effectively solved the problem of the slow maturation (7). Another approach to overcoming these shortcomings has been to continue the search for DsRed homologues in sea coral and anemone, an ...

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

Mitochondrial calcium uptake plays a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients, and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here, we utilize an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics, and organelle proteomics. RNA interference against 13 top candidates highlighted one gene that we now call mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the organelle’s inner membrane and has two canonical EF hands that are essential for its activity, suggesting a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high capacity mitochondrial calcium entry. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.

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