scholarly journals Single live-cell imaging for systems biology 9

2008 ◽  
Vol 45 ◽  
pp. 121-134 ◽  
Author(s):  
Dhanya Mullassery ◽  
Caroline A. Horton ◽  
Christopher D. Wood ◽  
Michael R.H. White

Understanding how mammalian cells function requires a dynamic perspective. However, owing to the complexity of signalling networks, these non-linear systems can easily elude human intuition. The central aim of systems biology is to improve our understanding of the temporal complexity of cell signalling pathways, using a combination of experimental and computational approaches. Live-cell imaging and computational modelling are compatible techniques which allow quantitative analysis of cell signalling pathway dynamics. Non-invasive imaging techniques, based on the use of various luciferases and fluorescent proteins, trace cellular events such as gene expression, protein–protein interactions and protein localization in cells. By employing a number of markers in a single assay, multiple parameters can be measured simultaneously in the same cell. Following acquisition using specialized microscopy, analysis of multi-parameter time-lapse images facilitates the identification of important qualitative and quantitative relationships–linking intracellular signalling, gene expression and cell fate.

2015 ◽  
Vol 166 (1-4) ◽  
pp. 101-103 ◽  
Author(s):  
M. Noguchi ◽  
Y. Kanari ◽  
A. Yokoya ◽  
A. Narita ◽  
K. Fujii

2014 ◽  
Vol 42 (11) ◽  
pp. e90-e90 ◽  
Author(s):  
Ilchung Shin ◽  
Judhajeet Ray ◽  
Vinayak Gupta ◽  
Muslum Ilgu ◽  
Jonathan Beasley ◽  
...  

2017 ◽  
Author(s):  
Noa Aloush ◽  
Tomer Schvartz ◽  
Andres I. König ◽  
Sarit Cohen ◽  
Eugene Brozgol ◽  
...  

ABSTRACTGenetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase/ pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNA contributes to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cells images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.


Author(s):  
Darren Machin ◽  
Daniel Williamson ◽  
Peter Fisher ◽  
victoria miller ◽  
Gemma Wildsmith ◽  
...  

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase-labelling approach to generate site-specifically <i>N</i>-terminally modified variants of both the A2-B<sub>5</sub> heterohexamer and B<sub>5</sub> pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B<sub>5</sub> pentamer showed an unexpected localization in the <i>medial/trans</i> Golgi. This study suggests a future role for specifically-labelled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labelling of lipid-rafts in fixed cells.


2006 ◽  
Vol 312 (4) ◽  
pp. 443-456 ◽  
Author(s):  
Horst Wolff ◽  
Kamyar Hadian ◽  
Manja Ziegler ◽  
Claudia Weierich ◽  
Susanne Kramer-Hammerle ◽  
...  

2014 ◽  
Vol 55 (suppl 1) ◽  
pp. i129-i130 ◽  
Author(s):  
Y. Kanari ◽  
M. Noguchi ◽  
K. Kaminaga ◽  
Y. Sakamoto ◽  
A. Yokoya

2015 ◽  
Vol 31 (11) ◽  
pp. 1816-1823 ◽  
Author(s):  
Theresa Niederberger ◽  
Henrik Failmezger ◽  
Diana Uskat ◽  
Don Poron ◽  
Ingmar Glauche ◽  
...  

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