Amy E. Palmer

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

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.

A monomeric red fluorescent protein

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 ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

Oxygen Binding, Activation, and Reduction to Water by Copper Proteins

Copper active sites play a major role in biological and abiological dioxygen activation. Oxygen intermediates have been studied in detail for the proteins and enzymes involved in reversible O(2) binding (hemocyanin), activation (tyrosinase), and four-electron reduction to water (multicopper oxidases). These oxygen intermediates exhibit unique spectroscopic features indicative of new geometric and electronic structures involved in oxygen activation. The spectroscopic and quantum-mechanical study of these intermediates has defined geometric- and electronic-structure/function correlations, and developed detailed reaction coordinates for the reversible binding of O(2), hydroxylation, and H-atom abstraction from different substrates, and the reductive cleavage of the O-O bond in the formation water.

MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake

Mitochondrial calcium uptake has 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 use 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, CBARA1, that we call hereafter 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 mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. 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.

Measuring calcium signaling using genetically targetable fluorescent indicators.

Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take ∼1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take ∼3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take ∼1 d depending on the extent of analysis.

Advances in fluorescence labeling strategies for dynamic cellular imaging

Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors

Zn2+ plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn2+ levels. Although much is known about total Zn2+ in cells, very little is known about its subcellular distribution. Yet defining the location of Zn2+ and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn2+ levels as well as flux of Zn2+ into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn2+ that is 100 times lower than the cytosol. Both organelles take up Zn2+ when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn2+ and thus have the potential to play a role in influencing Zn2+ toxicity. ER Zn2+ homeostasis is perturbed by small molecule antagonists of Ca2+ homeostasis and ER Zn2+ is released upon elevation of cytosolic Ca2+ pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca2+ signaling can influence Zn2+ homeostasis and vice versa, that Zn2+ dynamics may modulate Ca2+ signaling.

Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor.

Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brains responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type–specific expression. Fluorescent Ca2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo. Although suboptimal Ca2+-binding dynamics and Ca2+-induced fluorescence changes in FCIPs are important factors, low levels of expression also seem to play a role. Here we report that delivering D3cpv, an improved fluorescent resonance energy transfer–based FCIP, using a recombinant adeno-associated virus results in expression sufficient to detect the Ca2+ transients that accompany single action potentials. In upper-layer cortical neurons, we were able to detect transients associated with single action potentials firing at rates of <1 Hz, with high reliability, from in vivo recordings in living mice.

Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn2+ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn2+, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn2+ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn2+ is taken up into mitochondria following glutamate/Zn2+ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn2+ signals under different cellular conditions.

Fluorescent biosensors of protein function

Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.