Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals

Ohkura, Masamichi; Sasaki, Takuya; Sadakari, Junko; Gengyo-Ando, Keiko; Kagawa-Nagamura, Yuko; Kobayashi, Chiaki; Ikegaya, Yuji; Nakai, Junichi

doi:10.1371/journal.pone.0051286

Cited by 227 publications

(181 citation statements)

References 32 publications

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“…We next analyzed changes in [Ca 2+ ]i (intra-axonal Ca 2+ concentration) using a lentiviral vector for G-CaMP7, a genetically encoded Ca 2+ indicator. 30 We observed a substantial increase in [Ca 2+ ]i at 5 d.p.i. in the AxCANCre-infected Jsap1:Jlp dKO neurons, but not in single Jsap1 or Jlp KO or AxCANCreuninfected (control) Jsap1 f/f :Jlp f/f neurons (Figures 7a and b).…”

Section: Jsap1 and Jlp Regulate Axonal Transport T Sato Et Almentioning

confidence: 54%

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

et al. 2015

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Axonal transport is critical for neuronal development and function, and defective axonal transport has been implicated in neurodegenerative diseases. However, how axonal transport is regulated, or how defective transport leads to neuronal degeneration, remains unclear. Here, we report that c-Jun NH 2 -terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3 (JIP3)) and JNK-associated leucine zipper protein (JLP) are essential for postnatal brain development. Mice with a double-knockout (dKO) in Jsap1 and Jlp in the dorsal telencephalon developed progressive neuron loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JLP regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JLP to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. Furthermore, we describe a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jlp dKO neurons causes axonal degeneration and subsequent neuronal death. JNK hyperactivation because of increased intra-axonal Ca 2+ in the Jsap1:Jlp dKO neurons was found to mediate both the axonal degeneration and neuronal death, in cooperation with the Ca 2+ -dependent protease calpain. Our results indicate that axonal JNK may relocate to the nucleus in a dynein-dependent manner, where it activates the transcription factor c-Jun, resulting in neuronal death. Taken together, our data establish JSAP1 and JLP as positive regulators of kinesin-1-dependent axonal transport, which prevents neuronal degeneration.

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Section: Jsap1 and Jlp Regulate Axonal Transport T Sato Et Almentioning

confidence: 54%

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

et al. 2015

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“…45) and isotropic media using an in-phase/anti-phase (IPAP) HSQC experiment 46 .The spectra were processed using NMRpipe 47 and analyzed by SPARKY (http://www.cgl.ucsf.edu/home/sparky/) and CARA 48 . 1 H, 13 C and 15 N chemical shifts were calibrated indirectly by external DSS references.…”

Section: Methodsmentioning

confidence: 99%

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

et al. 2014

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Imaging with GECIs has become a widely used method in physiology and neuroscience [1][2][3] . According to readout mode, the design of the sensors has followed two different pathways, leading to single-wavelength sensors and FRET-based ratiometric sensors [4][5][6][7][8] . Among the most popular single-wavelength sensors are the G-CaMPs 9-13 , R-CaMPs 14 and GECOs 15 . FRET sensors include yellow cameleon 3.60 (refs. 16,17), D3cpv 18 , yellow cameleon Nano 19 and TN-XXL 20 .Quantification by ratiometric FRET imaging is more accurate than single-channel measurements and may be more suitable for long-term functional imaging studies, as it is less influenced by changes in optical path length, excitation light intensity and indicator expression level and by tissue movement and growth during development. In addition, FRET indicators are substantially brighter than single-wavelength sensors at rest, allowing better identification of expressing cells and their subcellular structures. Another practical feature of FRET-based indicators is their ability to measure basal Ca 2+ levels within cells, for example, to distinguish between resting and continuously spiking neuronssomething that cannot easily be achieved with single-wavelength indicators 21 . Increased basal Ca 2+ levels within the brain are also observed at the onset of neurodegenerative processes, and ratiometric FRET calcium imaging has been used in these conditions to monitor disease progression 22,23 . Moreover, ratiometric indicators are advantageous for monitoring calcium in motile cells.Both calmodulin and troponin C (TnC), the calcium binding proteins within the various GECIs, consist of two globular domains connected by a central linker 24,25 . Each domain possesses two calcium-binding EF hand motifs. Thus, currently available GECIs are highly nonlinear sensors binding up to four calcium ions per sensor. Identification of a smaller calciumbinding domain with fewer binding sites could help to reduce buffering during long-term chronic GECI expression 26 , make the sensor smaller and further minimize the risk of cytotoxicity. It may also help to simplify response properties and facilitate the biophysical modeling of sensor behavior.Here we report several improvements of FRET-based calcium sensors for in vivo imaging. First, we identified a minimal calcium binding motif based on the C-terminal domain of TnC with only two or one remaining calcium binding sites per sensor molecule, thus reducing the overall calcium buffering of the sensors. Second, by sampling TnCs from various species we identified a new TnC variant from the toadfish Opsanus tau, which offered the possibility of generating minimal domains with high-affinity calcium binding. Third, we used a large-scale, two-step functional screen to optimize the FRET changes in the sensor by linker diversification. This approach allowed us to identify Twitch sensors with a superior FRET change and may become useful for optimizing other types of FRET sensors. Finally, we improved brightness and photostability o...

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“…These changes, especially at the interfacial region where CaM interacts with cpGFP, allosterically alter the local environment of the tyrosine-derived chromophore and lead to dramatic fluorescence change in the presence of Ca 2+ (9,10). Because GCaMP and GECO proteins are genetically encodable, show sensitivity to physiologically relevant Ca 2+ concentrations, and respond to Ca 2+ concentration changes rapidly, they have gained increasing popularity for in vivo imaging of Ca 2+ in neural and olfactory cells (11)(12)(13).…”

mentioning

confidence: 99%

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

Oscar

Liu

Zhao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

191

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Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca 2+ ) sensing. This study reveals that, in the absence of Ca 2+ , the dominant skeletal motion is a ∼170 cm −1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca 2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca 2+ in physiologically relevant environments.calcium-sensing fluorescent protein | femtosecond Raman spectroscopy | fluorescence modulation mechanism | molecular movie G reen fluorescent protein (GFP) first emerged as a revolutionary tool for bioimaging and molecular and cellular biology about 20 years ago (1-3), and the quest to discover and engineer biosensors with improved and expanded functionality has yielded exciting advances. Recently, the color palette of genetically encoded Ca 2+ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4-7). The GECO proteins belong to the GCaMP family of Ca 2+ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8). The CaM unit undergoes large-scale structural changes upon Ca 2+ binding as it wraps around M13. These changes, especially at the interfacial region where CaM interacts with cpGFP, allosterically alter the local environment of the tyrosine-derived chromophore and lead to dramatic fluorescence change in the presence of Ca 2+ (9, 10). Because GCaMP and GECO proteins are genetically encodable, show sensitivity to physiologically relevant Ca 2+ concentrations, and respond to Ca 2+ concentration changes rapidly, they have gained increasing popularity for in vivo imaging of Ca 2+ in neur...

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Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals

Cited by 227 publications

References 32 publications

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

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