scholarly journals Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

2017 ◽  
Author(s):  
Noa Aloush ◽  
Tomer Schvartz ◽  
Andres I. König ◽  
Sarit Cohen ◽  
Eugene Brozgol ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Stop Codon ◽  
Signal To Noise Ratio ◽  
Trna Synthetase ◽  
Live Cell ◽  
Live Cells ◽  
Copy Numbers ◽  
Intracellular Proteins

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.

scholarly journals Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins

2016 ◽  
Vol 7 (1) ◽  
pp. 308-314 ◽  
Author(s):  
Yuko Kamikawa ◽  
Yuichiro Hori ◽  
Kazuo Yamashita ◽  
Lin Jin ◽  
Shinya Hirayama ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Modular Structure ◽  
Live Cells ◽  
Fluorogenic Probe ◽  
Intracellular Proteins ◽  
Protein Tag ◽  
Modular Structures

Quick and no-wash labeling of intracellular proteins was achieved in live cells using a PYP-tag mutant and a membrane-permeable fluorogenic probe with modular structures.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

scholarly journals Live Cell Imaging: 3-Dimensional Tracking of Non-blinking ‘Giant’ Quantum Dots in Live Cells (Adv. Funct. Mater. 30/2014)

2014 ◽  
Vol 24 (30) ◽  
pp. 4795-4795 ◽  
Author(s):  
Aaron M. Keller ◽  
Yagnaseni Ghosh ◽  
Matthew S. DeVore ◽  
Mary E. Phipps ◽  
Michael H. Stewart ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Live Cells ◽  
3 Dimensional

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

scholarly journals Label-free and live cell imaging by interferometric scattering microscopy

2018 ◽  
Vol 9 (10) ◽  
pp. 2690-2697 ◽  
Author(s):  
Jin-Sung Park ◽  
Il-Buem Lee ◽  
Hyeon-Min Moon ◽  
Jong-Hyeon Joo ◽  
Kyoung-Hoon Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Complex Structure ◽  
Live Cell ◽  
Fluorescent Label ◽  
Live Cells ◽  
Label Free ◽  
Cytoplasmic Organelles ◽  
Microscopic Techniques

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label.

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

2015 ◽  
Vol 166 (1-4) ◽  
pp. 101-103 ◽  
Author(s):  
M. Noguchi ◽  
Y. Kanari ◽  
A. Yokoya ◽  
A. Narita ◽  
K. Fujii
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Study ◽  
Live Cell ◽  
Mitochondrial Morphology ◽  
X Rays

scholarly journals PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs

2018 ◽  
Author(s):  
Livia V. Bayer ◽  
Omar S. Omar ◽  
Diana P. Bratu ◽  
Irina E. Catrina
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Beacon ◽  
Structural Information ◽  
Live Cell ◽  
Molecular Beacons ◽  
Intramolecular Interactions ◽  
Live Cells ◽  
Target Rna

ABSTRACTMolecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

scholarly journals Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

2019 ◽  
Author(s):  
Darren Machin ◽  
Daniel Williamson ◽  
Peter Fisher ◽  
victoria miller ◽  
Gemma Wildsmith ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Neuronal Tracing ◽  
Future Role ◽  
Golgi Localization

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.

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