Orange Fluorescent Proteins Research Articles

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins.

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 μM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM−1·cm−1 (DiLiFP561), 43.9 mM−1·cm−1 (DiLiFP570), and 31.2 mM−1·cm−1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime.Graphical abstract

Dipole Moment Variation Clears Up Electronic Excitations in the π-Stacked Complexes of Fluorescent Protein Chromophores.

We propose a quantitative structure-property relationship (QSPR) model for prediction of spectral tuning in cyan, green, orange, and red fluorescent proteins, which are engineered by motifs of the green fluorescent protein. Protein variants, in which their chromophores are involved in the π-stacking interaction with amino acid residues tyrosine, phenylalanine, and histidine, are prospective markers useful in bioimaging and super-resolution microscopy. In this work, we constructed training sets of the π-stacked complexes of four fluorescent protein chromophores (of the green, orange, red, and cyan series) with various substituted benzenes and imidazoles and tested the use of dipole moment variation upon excitation (DMV) as a descriptor to evaluate the vertical excitation energies in these systems. To validate this approach, we computed and analyzed electron density distributions of the π-stacked complexes and correlated the QSPR predictions with the reference values of the transition energies obtained using the high-level ab initio quantum chemistry methods. According to our results, the use of the DMV descriptor allows one to predict excitation energies in the π-stacked complexes with errors not exceeding 0.1 eV, which makes this model a practically useful tool in the development of efficient fluorescent markers for in vivo imaging.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

SummaryFibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Water-Soluble Flexible Organic Frameworks That Include and Deliver Proteins.

Four water-soluble hydrazone-based three-dimensional (3D) flexible organic frameworks FOF-1-4 have been synthesized from a semirigid tetracationic tetraaldehyde and four flexible dihydrazides. 1H NMR spectroscopy indicated the quantitative formation of FOF-1-4 in D2O, while dynamic light scattering experiments revealed that, depending on the concentration, these porous frameworks display hydrodynamic diameters ranging from 50 to 120 nm. The porosity of the frameworks is confirmed by ethanol vapor adsorption experiments of the solid samples as well as the high loading capacity for a 2.3 nm porphyrin guest in water. The new water-soluble frameworks exhibit low cytotoxicity and form inherent pores with diameters of 5.3 or 6.7 nm, allowing rapid inclusion of proteins such as bovine serum albumin and green and orange fluorescent proteins, and efficient delivery of the proteins into normal and cancer cells. Flow cytometric analysis reveals percentages of the delivered cells up to 99.8%.

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

SummaryParkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Chromatic Bacteria - A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria.

Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green–yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria–bacteria, bacteria–host, and bacteria–environment interactions.

Chemical cross-linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience.

Förster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and difficulty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green-emitting fluorescent proteins (FPs) as donors for a cross-linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., 46(5):516-522, 2018.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores.

Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.

Crystal Structure of Phototoxic Orange Fluorescent Proteins with a Tryptophan-Based Chromophore.

Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.

Multi-color imaging of the bacterial nucleoid and division proteins with blue, orange, and near-infrared fluorescent proteins.

Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5′-end of a gene strongly depends on the translational read-through of the inserted coding sequence.

Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues.

In vivo imaging of living organisms is an important tool to investigate biological phenomena. Two-photon excitation microscopy (2PEM) is a laser-scanning microscopy that provides noninvasive, deep imaging in living organisms based on the principle of multiphoton excitation. However, application of 2PEM to plant tissues has not been fully developed, as plant-specific autofluorescence, optically dense tissues, and multiple light-scattering structures diminish the clarity of imaging. In this study, the advantages of 2PEM were identified for deep imaging of living and intact Arabidopsis thaliana tissues. When compared to single-photon imaging, near-infrared 2PEM, especially at 1000 nm, reduced chloroplast autofluorescence; autofluorescence also decreased in leaves, roots, pistils, and pollen grains. For clear and deep imaging, longer excitation wavelengths using the orange fluorescent proteins (FPs) TagRFP and tdTomato gave better results than with other colors. 2PEM at 980 nm also provided multicolor imaging by simultaneous excitation, and the combination of suitable FPs and excitation wavelengths allowed deep imaging of intact cells in root tips and pistils. Our results demonstrated the importance of choosing both suitable FPs and excitation wavelengths for clear two-photon imaging. Further advances in in vivo analysis using 2PEM will facilitate more extensive studies in the plant biological sciences.

Orange Fluorescent Proteins: Filling the Spectral Gap

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.

Orange fluorescent proteins: structural studies of LSSmOrange, PSmOrange and PSmOrange2.

A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λex/λem at 437/572 nm), PSmOrange (λex/λem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λex/λem at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation was sufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution (Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiency of photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpected bathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structural changes associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For this reason the chromophore transformations accompanying photoconversion of the orange FPs are discussed.

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

Rabex-5 Protein Regulates Dendritic Localization of Small GTPase Rab17 and Neurite Morphogenesis in Hippocampal Neurons

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.

The mFruit Collection of Monomeric Fluorescent Proteins

Featured Article: Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004:22;1567–72.2 Fluorescent proteins, starting with the green fluorescent protein (GFP)3 from Aequorea victoria, have revolutionized our ability to noninvasively study living systems (1). Practically every cell biologist has used fluorescent proteins to tag a favorite protein and watch its dynamic localization in living cells. For almost 10 years after the gene encoding GFP was cloned, researchers were limited to using only about half of the visible spectrum with these probes and were able to image only 2 different colors simultaneously, at best. In 1999, Mikhail Matz and colleagues at the Russian Academy of Science discovered the first known red fluorescent protein, in Discosoma sp. coral; that protein would become known as DsRed (2). Hopes were high that the wavelength barrier had been broken, and many researchers jumped at the chance to try this new color. Unfortunately, it soon …

Development of New Fluorescent Voltage Sensor Proteins

Our goal is to develop genetically encoded fluorescent biosensors (FP voltage sensors) and to use them for optical recording of membrane potential in excitable cells. We created a series of FRET (fluorescence resonance energy transfer) based sensors. These constructs contained the voltage sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as voltage sensor fused with two fluorescent proteins, UKG (green-emitting fluorescent protein Umi-Kinoko) and mKOK (orange-emitting fluorescent protein Kusabira orange) as donor and acceptor. We used PCR to generate a library of constructs with insertions of the green and orange fluorescent proteins into different regions in CiVSP. Insertion of FRET donor and acceptor at different locations affects plasma membrane expression, FRET signal intensity, and response time constant. We are screening the these FP voltage sensors by examining the FRET signal in response to changes in membrane potential in HEK293 cells. Several novel probes have been identified; some with relatively large signals (>7% ΔF/F) and fast taus (<3 msec). We hope this effort will lead to voltage sensor proteins useful for in vivo recording of membrane potential in neurons.Supported by US NIH Grants DC005259 and NS054270 and grant WCI 2009-003 from the National Research Foundation of Korea.

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca2+ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca2+ ([Ca2+]cyto) can be excellently quantified with the ratiometric Ca2+ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca2+ sensors, the cameleons, are efficiently used to specifically measure Ca2+ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca2+]cyto and mitochondrial Ca2+ ([Ca2+]mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca2+ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca2+]cyto and [Ca2+]mito within same individual cells. Our data indicate that depending on the kinetics of [Ca2+]cyto rises there is a significant lag between onset of [Ca2+]cyto and [Ca2+]mito signals, pointing to a certain threshold of [Ca2+]cyto necessary to activate mitochondrial Ca2+ uptake. The temporal correlation between [Ca2+]mito and [Ca2+]cyto as well as the efficiency of the transfer of Ca2+ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca2+ extrusion and a desensitization of mitochondrial Ca2+ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca2+ oscillations of pancreatic beta-cells in response to D-glucose.