Repetitive Reversible Labeling of Proteins at Polyhistidine Sequences for Single-Molecule Imaging in Live Cells

ChemPhysChem ◽  
2007 ◽  
Vol 8 (8) ◽  
pp. 1221-1227 ◽  
Author(s):  
Emmanuel G. Guignet ◽  
Jean-Manuel Segura ◽  
Ruud Hovius ◽  
Horst Vogel
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

2008 ◽  
Vol 130 (29) ◽  
pp. 9204-9205 ◽  
Author(s):  
Samuel J. Lord ◽  
Nicholas R. Conley ◽  
Hsiao-lu D. Lee ◽  
Reichel Samuel ◽  
Na Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

Single-Molecule Imaging of RNA Splicing in Live Cells

2015 ◽  
pp. 571-585 ◽  
Author(s):  
José Rino ◽  
Robert M. Martin ◽  
Célia Carvalho ◽  
Ana C. de Jesus ◽  
Maria Carmo-Fonseca
Keyword(s):  
Single Molecule ◽  
Rna Splicing ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals Single Molecule Imaging of C9orf72 RNA and Repeat Associated Non-ATG Translation in Live Cells

2019 ◽  
Vol 116 (3) ◽  
pp. 299a
Author(s):  
Malgorzata J. Latallo ◽  
Shaopeng Wang ◽  
Nathan Livingston ◽  
Shuying Sun ◽  
Bin Wu
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals Single-Molecule Imaging of RNA in Live Cells

2016 ◽  
Vol 110 (3) ◽  
pp. 522a
Author(s):  
Maria Carmo-Fonseca
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals TALEN outperforms Cas9 in editing heterochromatin target sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Surbhi Jain ◽  
Saurabh Shukla ◽  
Che Yang ◽  
Meng Zhang ◽  
Zia Fatma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Gene Editing ◽  
Selective Recognition ◽  
Live Cells ◽  
Single Molecule Imaging ◽  
Local Searches ◽  
Search Mechanism ◽  
Efficient Gene ◽  
Target Sites

AbstractGenome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.

scholarly journals Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

2012 ◽  
Vol 103 (8) ◽  
pp. 1691-1697 ◽  
Author(s):  
Jinwoo Lee ◽  
Yukihiro Miyanaga ◽  
Masahiro Ueda ◽  
Sungchul Hohng
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals Differential Mast Cell Outcomes Are Sensitive to FcεRI-Syk Binding Kinetics

2017 ◽  
Author(s):  
Samantha L. Schwartz ◽  
Cédric Cleyrat ◽  
Mark Olah ◽  
Peter Relich ◽  
Genevieve Phillips ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Calcium Release ◽  
Aggregate Size ◽  
Live Cells ◽  
Single Molecule Imaging ◽  
Site Specific ◽  
Sh2 Domains ◽  
Downstream Signaling ◽  
Transient Nature

AbstractCrosslinking of IgE-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.SummarySchwartz et al. use single molecule imaging to quantify the transient nature of FcεRI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

scholarly journals Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging

2021 ◽  
Author(s):  
Thomas George Wade Graham ◽  
John Joseph Ferrie ◽  
Gina M. Dailey ◽  
Robert Tjian ◽  
Xavier Darzacq
Keyword(s):  
Single Molecule ◽  
Molecular Interactions ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Single Molecule Imaging ◽  
Protein Protein Interactions ◽  
Self Association

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the "sender") can reactivate a second fluorophore (the "receiver") from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Forster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

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