ABSTRACT
StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.
Subject(s)
Light , Lighting , Humans , HeLa Cells , Coloring Agents , Microscopy, ConfocalABSTRACT
Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. We applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.
Subject(s)
Golgi Apparatus , Mitochondria , Mitochondria/chemistry , Golgi Apparatus/metabolism , Microtubules/metabolism , Microscopy, Confocal/methodsABSTRACT
The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.
Subject(s)
COVID-19 , Endoplasmic Reticulum , Green Fluorescent Proteins/genetics , Humans , Microscopy, Fluorescence/methodsABSTRACT
Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
Subject(s)
Luciferases, Firefly/chemistry , Luminescent Measurements/methods , Neurons/cytology , Single-Cell Analysis/methods , Animals , Benzothiazoles/chemistry , Callithrix , Carcinogenesis/chemistry , Carcinogenesis/pathology , Corpus Striatum/chemistry , Corpus Striatum/cytology , Directed Molecular Evolution , Hippocampus/chemistry , Luciferases, Firefly/genetics , Lung/blood supply , Mice , Movement , Neurons/chemistry , Protein Engineering , Video RecordingABSTRACT
In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid (RA) is an important morphogen involved in patterning the anterior-posterior axis of structures, including the hindbrain and paraxial mesoderm. RA diffuses over long distances, and its activity is spatially restricted by synthesizing and degrading enzymes. However, gradients of endogenous morphogens in live embryos have not been directly observed; indeed, their existence, distribution and requirement for correct patterning remain controversial. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded probes for RA). Using the principle of fluorescence resonance energy transfer we engineered the ligand-binding domains of RA receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor and acceptor, respectively, for the reliable detection of ambient free RA. We created three GEPRAs with different affinities for RA, enabling the quantitative measurement of physiological RA concentrations. Live imaging of zebrafish embryos at the gastrula and somitogenesis stages revealed a linear concentration gradient of endogenous RA in a two-tailed source-sink arrangement across the embryo. Modelling of the observed linear RA gradient suggests that the rate of RA diffusion exceeds the spatiotemporal dynamics of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients to tissue engineering and regenerative medicine.
Subject(s)
Embryonic Development/physiology , Tretinoin/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Body Patterning/physiology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Fluorescence Resonance Energy Transfer , Gastrula/embryology , Gastrula/metabolism , HeLa Cells , Humans , Models, Biological , Molecular Probes/analysis , Molecular Probes/genetics , Molecular Probes/metabolism , Molecular Sequence Data , Rhombencephalon/embryology , Rhombencephalon/metabolism , Somites/embryology , Somites/metabolism , Substrate Specificity , Tretinoin/analysis , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The nuclear envelope (NE) defines the nuclear compartment, and nuclear pore complexes (NPCs) on the NE form aqueous passages through which small water-soluble molecules can passively diffuse. It is well known that proteins smaller than 50 kDa can diffuse though NPCs, whereas proteins larger than 60 kDa rarely enter by passive diffusion. Little, however, is known about how this size cutoff develops as the NE reassembles and the nucleus expands. In 1987, a well-known study identified an efficient mechanism by which large diffusing proteins (> 60 kDa) were excluded from the reassembling nucleus after mitosis. Since then, it has been generally accepted that after mitosis, newly formed nuclei completely exclude all proteins except those that are initially bound to the mitotic chromosomes and those that are selectively imported through NPCs. Here, the tetrameric complex of the photoconvertible fluorescent protein KikGR ( approximately 103 kDa) was optically highlighted in the cytoplasm and followed to examine its entry into nuclei. Remarkably, highlighted complexes efficiently entered newly assembled nuclei during an approximately 20-min period after the completion of cytokinesis. Because KikGR contains no known nuclear-localization or chromosome-binding sequences, our results indicate the diffusion barrier is less restrictive during nuclear reassembly.
Subject(s)
Cell Division/physiology , Cell Nucleus/metabolism , Green Fluorescent Proteins/metabolism , Optics and Photonics/methods , Active Transport, Cell Nucleus , Animals , Anthozoa , Cell Membrane Permeability , Cytokinesis/physiology , Cytoplasm/metabolism , Diffusion , Fluorescence , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Mitosis , Models, Molecular , Mutation , Time FactorsABSTRACT
For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.
Subject(s)
Calcium/metabolism , Fluorescence Polarization/methods , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Algorithms , Animals , Calcium/analysis , Calcium Signaling/physiology , Fluorescence Polarization/instrumentation , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentationABSTRACT
Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Here we discuss how to engineer constructs for FRET between the cyan- and yellow-emitting variants of green fluorescent protein (GFP) from Aequorea victoria (CFP and YFP, respectively). Throughout this chapter, we stress the fact that FRET is highly sensitive to the relative orientation and distance between the donor and the acceptor. The chapter consists of two parts. First, we discuss FRET-based indicators encoded by single genes, which were developed in our laboratory. In this approach, a number of different constructs can be made for a comparative assessment of their FRET efficiencies. For example, the length and sequence of the linker between the fluorescent protein and the host protein should be optimized for each specific application. In the second part, we describe the use of long and flexible linkers for engineering FRET constructs, including an introduction to a general and efficient tool for making successful fusion proteins with long and flexible linkers. When CFP and YFP are fused through floppy linkers to two protein domains that interact with each other, the two fluorescent proteins will associate due to the weak dimerization propensity of Aequorea GFP, which results in moderate FRET. This approach has become even more powerful due to the construction of a new pair of fluorescent proteins for FRET: CyPet and YPet.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins , Luminescent Proteins , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Recombinant Fusion Proteins , Base Sequence , HeLa Cells , Humans , Molecular Sequence Data , TransfectionABSTRACT
Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
Subject(s)
Calcium/metabolism , DNA, Mitochondrial/genetics , Hydrogen-Ion Concentration , Parkinson Disease/genetics , Polymorphism, Genetic , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , HumansABSTRACT
Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.
Subject(s)
Bacterial Proteins/chemistry , Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemistry , Anisotropy , Bacterial Proteins/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Kinetics , Lasers , Luminescent Proteins/metabolism , Peptide Hydrolases/metabolism , Peptide Hydrolases/pharmacology , Photobleaching , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Dual-excitation ratiometric dyes permit quantitative measurements of Ca2+ concentrations ([Ca2+]s), by minimizing the effects of several artifacts that are unrelated to changes in [Ca2+]. These dyes are excited at two different wavelengths, and the resultant fluorescence intensities are measured sequentially. Therefore, it is difficult to follow fast [Ca2+] dynamics or [Ca2+] changes in highly motile cell samples. To overcome this problem, we have developed a new dual-excitation ratiometry system that employs two high-power light-emitting diodes (LEDs), two high-speed liquid crystal shutters, and a CCD camera. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity. We demonstrate the effectiveness of this system by monitoring changes in [Ca2+] in cardiac muscle cells loaded with Fura-2.
Subject(s)
Calcium/metabolism , Fura-2/administration & dosage , Image Enhancement/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Myocytes, Cardiac/metabolism , Semiconductors , Signal Processing, Computer-Assisted , Animals , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Image Enhancement/methods , Lighting , Liquid Crystals , Microscopy, Fluorescence, Multiphoton/methods , Myocytes, Cardiac/drug effects , RatsABSTRACT
The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding. We used a new ratiometric fluorescent calcium indicator and a new optical system that allows simultaneous illumination and detection at any two wavelengths. Pumping was observed with fast, repetitive and synchronous spikes in calcium concentrations in the corpus and terminal bulb, indicative of electrical coupling throughout the muscles. The posterior isthmus, however, responded to only one out of several pumping spikes to produce broad calcium transients, leading to peristalsis, the slow and gradual motion needed for efficient swallows. The excitation-calcium coupling may be uniquely modulated in this region at the level of calcium channels on the plasma membrane.
Subject(s)
Caenorhabditis elegans/physiology , Calcium/metabolism , Muscle Contraction , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Muscles/physiology , Peristalsis , Pharynx , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
Dual-excitation ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]). These dyes are excited alternately at two different wavelengths, and the pair of intensity measurements must be collected sequentially. Therefore, it is difficult to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples. Here, we present a novel but simple dual-excitation ratiometric method which overcomes this problem. By the use of our home-made illuminator, each sample is illuminated by two orthogonal linear polarized lights of different wavelengths. Fluorescence images are captured by two CCD cameras through two analyzers, whose polarization directions are at right angles. This methodology allows us to perform simultaneous measurements of any dual-excitation ratiometric dye, and we demonstrate its validity by monitoring [Ca2+] changes in rat cardiac muscle cells loaded with Fura Red.
Subject(s)
Calcium/analysis , Microscopy, Fluorescence/methods , Myocytes, Cardiac/metabolism , Animals , Calcium/metabolism , Calcium Signaling , Fluorescence Polarization/instrumentation , Fluorescence Polarization/methods , Fluorescent Dyes/chemistry , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Microscopy, Fluorescence/instrumentation , Myocytes, Cardiac/cytology , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
The procerebrum (PC) of the terrestrial slug shows a coherent oscillatory activity. Information is encoded in the PC by neurons with synchronized oscillatory activity, and the oscillatory activity will propagate to other brain regions as the PC transmits information. Using the optical recording of membrane potentials and the correlation analysis, we showed that the metacerebrum/mesocerebrum (MC) region also shows an oscillatory activity coherent with that of the PC. The MC oscillation was either inphase or antiphase with the PC oscillation, and its amplitude was larger when it was antiphase than it was inphase. These results indicate that the MC is capable of producing an oscillatory activity, possibly driven by synaptic input from the PC.
Subject(s)
Action Potentials/physiology , Biological Clocks/physiology , Brain Mapping/methods , Microscopy, Fluorescence/methods , Mollusca/physiology , Nerve Net/physiology , Neurons/physiology , Telencephalon/physiology , Animals , Image Interpretation, Computer-Assisted/methods , Membrane Potentials/physiology , Olfactory Receptor Neurons/physiology , Statistics as TopicABSTRACT
Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca(2+) imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca(2+) using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca(2+) waves from the cytosol to the nucleus and changes in Ca(2+) concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.