scholarly journals Fast and sensitive GCaMP calcium indicators for imaging neural populations

2021 ◽  
Author(s):  
Loren Looger ◽  
Yan Zhang ◽  
Martón Rózsa ◽  
Yajie Liang ◽  
Daniel Bushey ◽  
...  
Keyword(s):  
Neural Activity ◽  
Calcium Binding ◽  
Large Scale ◽  
Fast Kinetics ◽  
Trade Offs ◽  
Calcium Indicators ◽  
Electrical Signaling ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Large Scale Screening

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems. The popular GCaMP indicators are based on the calcium-binding protein calmodulin and the RS20 peptide. These sensors report neural activity at timescales much slower than electrical signaling, limited by their biophysical properties and trade-offs between sensitivity and speed. We used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting jGCaMP8 sensors, based on calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (rise times, 2 ms) and still feature the highest sensitivity for neural activity reported for any protein-based sensor. jGCaMP8 sensors will allow tracking of larger populations of neurons on timescales relevant to neural computation.

scholarly journals Fast and sensitive GCaMP calcium indicators for imaging neural populations

2021 ◽  
Author(s):  
Loren Looger ◽  
Yan Zhang ◽  
Martón Rózsa ◽  
Yajie Liang ◽  
Daniel Bushey ◽  
...  
Keyword(s):  
Neural Activity ◽  
Calcium Binding ◽  
Large Scale ◽  
Fast Kinetics ◽  
Trade Offs ◽  
Calcium Indicators ◽  
Electrical Signaling ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Large Scale Screening

Abstract Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems. The popular GCaMP indicators are based on the calcium-binding protein calmodulin and the RS20 peptide. These sensors report neural activity at timescales much slower than electrical signaling, limited by their biophysical properties and trade-offs between sensitivity and speed. We used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting ‘jGCaMP8’ sensors, based on calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (rise times, 2 ms) and still feature the highest sensitivity for neural activity reported for any protein-based sensor. jGCaMP8 sensors will allow tracking of larger populations of neurons on timescales relevant to neural computation.

scholarly journals Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase

2019 ◽  
Vol 295 (8) ◽  
pp. 2203-2211 ◽  
Author(s):  
Joseph J. Porter ◽  
Hyo Sang Jang ◽  
Mohammad Mahfuzul Haque ◽  
Dennis J. Stuehr ◽  
Ryan A. Mehl
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Calcium Binding ◽  
Vascular Dysfunction ◽  
Post Translational Modification ◽  
Calcium Dependent ◽  
Genetic Code Expansion ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr–calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

scholarly journals Extra-Nuclear Functions of the Transcription Factor Grainyhead-Like 3 in the Endothelium—Interaction with Endothelial Nitric Oxide Synthase

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 428
Author(s):  
Kirsten Jander ◽  
Jan Greulich ◽  
Stefanie Gonnissen ◽  
Niloofar Ale-Agha ◽  
Christine Goy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Transcription Factor ◽  
Nitric Oxide Synthase ◽  
Transcriptional Activation ◽  
Endothelial Nitric Oxide Synthase ◽  
Large Scale ◽  
Deletion Mutants ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

We previously demonstrated that the transcription factor Grainyhead-like 3 (GRHL3) has essential functions in endothelial cells by inhibiting apoptosis and promoting migration as well as activation of endothelial nitric oxide synthase (eNOS). We now show that a large portion of the protein is localized to myo-endothelial projections of murine arteries suggesting extra-nuclear functions. Therefore, we generated various deletion mutants to identify the nuclear localization signal (NLS) of GRHL3 and assessed potential extra-nuclear functions. Several large-scale deletion mutants were incapable of activating a GRHL3-dependent reporter construct, which could either be due to deficiencies in transcriptional activation or to impaired nuclear import. One of these mutants encompassed a predicted bipartite NLS whose deletion led to the retention of GRHL3 outside the nucleus. Interestingly, this mutant retained functions of the full-length protein as it could still inhibit pathways inducing endothelial cell apoptosis. As apoptosis protection by GRHL3 depends on NO-production, we examined whether GRHL3 could interact with eNOS and showed a direct interaction, which was enhanced with the extra-nuclear GRHL3 variant. The observation that endogenous GRHL3 also interacts with eNOS in intact murine arteries corroborated these findings and substantiated the notion that GRHL3 has important extra-nuclear functions in the endothelium.

scholarly journals High-Throughput Genotyping with Single Nucleotide Polymorphisms

2001 ◽  
Vol 11 (7) ◽  
pp. 1262-1268
Author(s):  
Koustubh Ranade ◽  
Mau-Song Chang ◽  
Chih-Tai Ting ◽  
Dee Pei ◽  
Chin-Fu Hsiao ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
High Throughput ◽  
Large Scale ◽  
Association Studies ◽  
Nucleotide Polymorphisms ◽  
Allelic Discrimination Assay ◽  
Allelic Discrimination ◽  
Single Nucleotide ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5′ nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11–β hydroxylase gene. The genotyping method is accurate—we estimate an error rate of fewer than 1 in 2000 genotypes, rapid—with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible—a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three “pseudo-SNPs” (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

scholarly journals Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39851 ◽  
Author(s):  
Pei-Rung Wu ◽  
Cheng-Chin Kuo ◽  
Shaw-Fang Yet ◽  
Jun-Yang Liou ◽  
Kenneth K. Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Calcium Binding ◽  
Endothelial Nitric Oxide Synthase ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Localization of endothelial nitric oxide synthase in the normal and failing human atrial myocardium

1996 ◽  
Vol 54 ◽  
pp. 786-787
Author(s):  
Chi-Ming Wei ◽  
Margarita Bracamonte ◽  
Shi-Wen Jiang ◽  
Richard C. Daly ◽  
Christopher G.A. McGregor ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cardiac Tissues ◽  
Mammalian Tissues ◽  
End Stage Heart Failure ◽  
End Stage ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Nitric oxide (NO) is a potent endothelium-derived relaxing factor which also may modulate cardiomyocyte inotropism and growth via increasing cGMP. While endothelial nitric oxide synthase (eNOS) isoforms have been detected in non-human mammalian tissues, expression and localization of eNOS in the normal and failing human myocardium are poorly defined. Therefore, the present study was designed to investigate eNOS in human cardiac tissues in the presence and absence of congestive heart failure (CHF).Normal and failing atrial tissue were obtained from six cardiac donors and six end-stage heart failure patients undergoing primary cardiac transplantation. ENOS protein expression and localization was investigated utilizing Western blot analysis and immunohistochemical staining with the polyclonal rabbit antibody to eNOS (Transduction Laboratories, Lexington, Kentucky).

Training Aides to Screen Children for Speech and Language Problems

1976 ◽  
Vol 7 (4) ◽  
pp. 236-241 ◽  
Author(s):  
Marisue Pickering ◽  
William R. Dopheide
Keyword(s):  
Rural Areas ◽  
Large Scale ◽  
School Personnel ◽  
School Age Children ◽  
Speech And Language ◽  
Elementary School Age ◽  
Evaluation Procedures ◽  
Language Problems ◽  
Competency Based ◽  
Large Scale Screening

This report deals with an effort to begin the process of effectively identifying children in rural areas with speech and language problems using existing school personnel. A two-day competency-based workshop for the purpose of training aides to conduct a large-scale screening of speech and language problems in elementary-school-age children is described. Training strategies, implementation, and evaluation procedures are discussed.

Sign in / Sign up

Export Citation Format

Share Document