scholarly journals Development of a genetically encoded sensor for endogenous CaMKII activity

2018 ◽  
Author(s):  
Goli Ardestani ◽  
Megan West ◽  
Thomas J. Maresca ◽  
Rafael A. Fissore ◽  
Margaret M. Stratton
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Linker Region ◽  
Fresh Perspective ◽  
Differential Responses ◽  
Mouse Eggs

ABSTRACTCaMKII is a crucial oligomeric enzyme in neuronal and cardiac signaling, fertilization and immunity. Here, we report the construction of a novel, substrate-based, genetically-encoded sensor for CaMKII activity, FRESCA (FRET-based Sensor for CaMKII Activity). Currently, there is one biosensor for CaMKII activity, Camui, which contains CaMKII. FRESCA allows us to measure all endogenous CaMKII variants, while Camui can track a single variant. Since there are ~40 CaMKII variants, using FRESCA to measure aggregate activity allows a fresh perspective on CaMKII activity. We show, using live-cell imaging, FRESCA response is concurrent with Ca2+ rises in HEK293T cells and mouse eggs. In eggs, we stimulate oscillatory patterns of Ca2+ and observe the differential responses of FRESCA and Camui. Our results implicate an important role for the variable linker region in CaMKII, which tunes its activation. FRESCA will be a transformative tool for studies in neurons, cardiomyocytes and other CaMKII-containing cells.

Simple Fluorescence Turn-On Chemosensor for Selective Detection of Ba2+ Ion and Its Live Cell Imaging

2019 ◽  
Vol 91 (15) ◽  
pp. 10095-10101 ◽  
Author(s):  
Palanisamy Ravichandiran ◽  
Sivakumar Allur Subramaniyan ◽  
Antony Paulraj Bella ◽  
Princy Merlin Johnson ◽  
Ae Rhan Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Selective Detection ◽  
Turn On ◽  
Fluorescence Turn On

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

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