Synthesis of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability (original) (raw)
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Fluorescence imaging of calcium ions (Ca 2+) has become an essential technique for investigation of sig-naling pathways involving Ca 2+ as a second messenger. But, Ca 2+ signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca 2+ and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca 2+ that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca 2+ concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca 2+ red-fluorescent probe showed high cell-membrane per-meability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.
New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2014
a b s t r a c t Q5 1 5 a r t i c l e i n f o j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b b a m c r Please cite this article as: M. Oheim, et al., New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging, Biochim. Biophys. Acta (2014), http://dx.the molar extinction ε and/or fluorescence quantum yield Φ F varies as a 66 function of Ca 2+ binding. These non-ratiometric single-wavelength 67 Ca 2+ indicators report relative fluorescence changes (F/F 0 or ΔF/F 0 ) 68 only and constitute the largest group. The advantages and disadvan-69 tages of each group have been discussed. It is safe to say that precise 70 measurements of basal cytoplasmic [Ca 2 + ] i values around 100 nM 71 are at best difficult [14-16], and whereas earlier studies often sought 72 to determine absolute [Ca 2 + ] i levels, the more recent literature is 73 dominated mostly by ΔF/F 0 (and, for some applications dual-color 74 green (G)/red (R) pseudoratiometric ΔG/R measurements with a 75 Ca 2 + -insensitive reference dye) measurements of Ca 2+ -dependent 76 fluorescence transients. 77 1.1. Working principle of chemical fluorescent Ca 2+ indicators 78 Chemical Ca 2+ indicators consist of a fluorophore moiety determin-79 ing their photophysical properties and an ionophore moiety complexing 80 Ca 2+ . Many, but not all indicators have a molecular linker between 81 these two parts (see below). Ca 2+ binding modifies the fluorescence 82 of the construct. Ca 2 + sensing is generally based on one of two 83 photophysical principles: photoinduced electron transfer (PET) or 84 photoinduced charge transfer (PCT) [17]. 85 The vast majority of Ca 2+ indicators are PET-type sensors [18]. PET 86 sensors combine an electron-donating group (D), which is part of the 87 Ca 2+ chelating moiety (an aniline in the case of BAPTA) that connects 88 to the conjugated aromatic system of the fluorescent dye through a 89 bridge. The donor group quenches fluorescence due to the electron 90 transfer from its highest occupied molecular orbital (HOMO) to the 91 'half-filled' HOMO of the excited fluorophore, which has a lower energy.
Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging
Cold Spring Harbor protocols, 2013
Imaging Ca(2+)-sensitive fluorescent indicators provides a common approach for studying Ca(2+) signals in many contexts. Fluorescent indicators are particularly useful for measuring acute Ca(2+) changes in a relatively noninvasive manner. The availability of indicators that can be targeted to specific cellular domains, coupled with variations in affinity, brightness or spectral characteristics, provides tools for exploring spatially and temporally diverse Ca(2+) signals, and moreover, multiplexing the readout of Ca(2+) with other cellular functions. This article aims to give the novice experimentalist some insight into the considerations and potential pitfalls that impinge on the use of fluorescent Ca(2+) indicators.
Loading fluorescent Ca2+ indicators into living cells
Cold Spring Harbor protocols, 2013
Small-molecule fluorescent Ca(2+) reporters are the most widely used tools in the field of Ca(2+) signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca(2+) as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca(2+) indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca(2+) indicators have carboxylic acid groups for binding of Ca(2+). Because these "free-acid" forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxyme...
Monitoring Intracellular Ca2+ in Brain Slices with Fluorescent Indicators
Molecular Biology Intelligence Unit
I maging fluorescent chemical indicators specific for calcium (Ca^*) has provided important insights into our current understanding of the many Ca^* regulated cellular processes in the brain such as neurotransmitter release and synaptic plasticity. In this chapter we discuss the use of fluorescent Ca * indicators for the measurement of intracellular concentrations ([Ca^^li) in brain slices. Single-wavelength intensity-modulating indicators are contrasted with dual-wavelength ratiometric indicators and high versus low-affinity indicators are described. The advantages and disadvantages of using a particular indicator form (free acid, AM-ester or dextran conjugate) for reliable Ca^* imaging are outlined. Finally, we review calibration methods to estimate intracellular [Ca^*] from both nonratiometric and ratiometric indicators. This chapter should provide a guide to how and when to use various Ca^* sensitive fluorescent indicators to map the spatio-temporal dynamics of intracellular [Ca *] in brain slices. Ca * Sensitive Fluorescent Chemical Indicators Our current understanding of the numerous Ca^* regulated physiological cellular phenomena in the brain has been greatly facilitated by the use of the Ca * sensitive fluorescent chemical probes developed by Tsien and colleagues. ^'^^ The most widely used fluorescent indicators for intracellular measurement of free Ca * concentration ([Ca *]i) are based on the Ca^* chelator l,2-bis-(2-aminophenoxy)ethane-A^,7V,7V_,A^_-tetraacetic acid (BAPTA) (Fig. 1). BAPTA has high selectivity for Ca^^ (Ka « lOOnM at pH 7.0) over competing concentrations of Mg^* and an extremely fast on rate (10^-lO^M'^S'^) for Ca^* binding. The main Ca * sensitive fluorescent indicators are obtained by coupling different fluorophores with varying spectral properties to the Ca^* sensor BAPTA. The binding of Ca^* to these Ca * sensitive indicators alters the excitation or emission spectra such that the fluorescence of the indicator that binds the Ca^* can be easily distinguished from the fluorescence of the indicator that remains Ca^* free. The most useful fluorescent probes are those with large molar extinction coefficients and quantum yields that exhibit strong and stable fluorescence well above any background tissue autofluorescence. The wide range of Ca * sensitive indicators now available (www.probes.com) can be divided into several operational classes based on a number of criteria, the advantages and disadvantages of which should be considered when selecting a probe for a particular experiment; 1) single-wavelength intensity-modulating probes vs. dual-wavelength ratiometric probes, 2) Ca^* binding affinity, and 3) indicator form (salt, AM ester or dextran conjugate). Voltage-Gated Calcium Channels^ edited by Gerald Zamponi.
Twenty years of calcium imaging: cell physiology to dye for
2005
The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca 2+ in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca 2+ signaling is accomplished. In excitable cells, the coordination of changing Ca 2+ concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca 2+ signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.
Nanoparticle PEBBLE Sensors for Quantitative Nanomolar Imaging of Intracellular Free Calcium Ions
Analytical Chemistry, 2012
Ca 2+ is a universal second messenger and plays a major role in intracellular signaling, metabolism and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca 2+ measurement involves introduction of optically sensitive Ca 2+ indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca 2+ indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most sensitive indicators, cytotoxicity of the indicators, interference from non-specific binding caused by cellular biomacromolecules, challenging calibration and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca 2+ indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine based Ca 2+ fluorescence indicators. The here presented rhod-2 containing PEBBLEs show a stable sensing range at near-neutral pH (pH 6-9). Due to the protection of the PEBBLE matrix, the interference of protein non-specific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K d = 478 nM) and intracellular (K d = 293 nM) measurements. These nanosensors are a useful quantitative tool for the measurement and imaging of the cytosolic nanomolar free Ca 2+ levels.
Scientific Reports, 2016
Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophorebased sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca 2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca 2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca 2+binding sites. The engineered sensor, called NTnC, uses TnC as the Ca 2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca 2+. Using NTnC, we have visualized Ca 2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca 2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca 2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope. Optical techniques using genetically encoded calcium indicators (GECIs) based on fluorescent proteins (FPs) are broadly applied for in vivo visualization of neuronal activity. FP-based calcium indicators (or sensors) can be classified into two major designs (Fig. 1a). The first class of GECIs includes the FRET (fluorescence resonance energy transfer)-based family of sensors, which is composed of two fluorescent proteins, one acting as a donor and another as an acceptor, with a Ca 2+-binding domain located between them 1. The latter can be represented by calmodulin (CaM) in combination with the M13 peptide from myosin light chain kinase (CaM/M13) or by a minimal Ca 2+-binding motif from the C-terminal domain of troponin C (TnC). In the first type of FRET sensor, CaM carries four calcium ion-binding
Bioinspired design of a polymer gel sensor for the realization of extracellular Ca2+ imaging
Scientific Reports, 2016
Although the role of extracellular Ca 2+ draws increasing attention as a messenger in intercellular communications, there is currently no tool available for imaging Ca 2+ dynamics in extracellular regions. Here we report the first solid-state fluorescent Ca 2+ sensor that fulfills the essential requirements for realizing extracellular Ca 2+ imaging. Inspired by natural extracellular Ca 2+-sensing receptors, we designed a particular type of chemically-crosslinked polyacrylic acid gel, which can undergo single-chain aggregation in the presence of Ca 2+. By attaching aggregation-induced emission luminogen to the polyacrylic acid as a pendant, the conformational state of the main chain at a given Ca 2+ concentration is successfully translated into fluorescence property. The Ca 2+ sensor has a millimolar-order apparent dissociation constant compatible with extracellular Ca 2+ concentrations, and exhibits sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically relevant ions, thus enabling monitoring of submillimolar fluctuations of Ca 2+ in flowing analytes containing millimolar Ca 2+ concentrations. Ca 2+ plays a crucial role in many important physiological and pathological processes in animals 1-17 and plants 9,18-23. Over the past several decades, many synthetic molecular and genetically encoded fluorescent Ca 2+ indicators have been developed, as represented by 1,2-bis(o-aminophenoxy)-ethane-N,N,N′ ,N′-tetraacetic acid (BAPTA) derivatives 24-27 and calmodulin-based proteins 28-32 , respectively. Ca 2+-imaging techniques that use such fluorescent indicators are indispensable in modern biology and medical science. In living organisms, Ca 2+ concentrations differ greatly depending on the compartment. Typically, the Ca 2+ concentration is ~100 nanomolar (nM) in intracellular cytosol, ~100 micromolar (μM) in the endoplasmic reticulum and mitochondria and ~1 millimolar (mM) in extracellular fluid and blood (Fig. 1a,b) 3. Plant vacuoles are also considered to contain mM-order Ca 2+ concentrations 20. Hence, Ca 2+ imaging in all of these compartments requires dedicated fluorescent indicators with specific dissociation constants (K d) that are appropriate for the respective background Ca 2+ concentrations. However, almost every Ca 2+ indicator known to date has a K d value ranging from nM to μM, and therefore allows for Ca 2+ imaging only in cytosol and organelles (Fig. 1a). Fluorescent Ca 2+ indicators with mM-order K d , compatible with extracellular Ca 2+ concentrations 27,32 , have scarcely been developed 9,10 , despite the fact that extracellular Ca 2+ , which is conventionally regarded as a diagnostic indicator for many diseases 3,7 , is now receiving considerable attention as a first messenger 3-17 in, for example, parathyroid gland 3,4 , neuron 12,13 , myocyte 14 , stem cell 15 and macrophages 16,17. In fact, there are major problems in the development of indicators for extracellular Ca 2+ imaging 9,10. First, such indicators should be designed to strike a balance between mM-order K d (i.e., a rather small affinity for Ca 2+) and high selectivity for Ca 2+ in the presence of excessive amounts of other physiological ions. Although simple Ca 2+ imaging against mM-order background concentration of Ca 2+ may be possible using existing indicators with μM-order K d , Ca 2+ indicators with one-order higher K d have a great advantage in monitoring Ca 2+ transients and oscillations in extracellular regions. Even more challenging in extracellular Ca 2+ imaging, one has to create a mechanism to avoid the outflow of indicators from an observation area through molecular diffusion. Obviously, this issue is intractable with existing molecular-based indicators.