scholarly journals Single-Molecule Tracking Approaches to Protein Synthesis Kinetics in Living Cells

Biochemistry ◽  
2018 ◽  
Vol 58 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Ivan L. Volkov ◽  
Magnus Johansson
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Tracking

Single-Molecule Tracking of mRNA in Living Cells

Nanoimaging ◽  
2012 ◽  
pp. 153-167
Author(s):  
Mai Yamagishi ◽  
Yoshitaka Shirasaki ◽  
Takashi Funatsu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Tracking

scholarly journals A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

2017 ◽  
Author(s):  
Rajarshi P Ghosh ◽  
J Matthew Franklin ◽  
Will E. Draper ◽  
Quanming Shi ◽  
Jan T. Liphardt
Keyword(s):  
Single Molecule ◽  
Precision Measurement ◽  
Single Molecules ◽  
Living Cells ◽  
Background Suppression ◽  
Molecular Heterogeneity ◽  
Single Molecule Tracking ◽  
High Brightness ◽  
Cellular Processes ◽  
State Switching

AbstractCellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

scholarly journals Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells

2021 ◽  
Vol 118 (5) ◽  
pp. e2012229118
Author(s):  
Zhongwen Chen ◽  
Yuhong Cao ◽  
Chun-Wei Lin ◽  
Steven Alvarez ◽  
Dongmyung Oh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Delivery ◽  
Chemical Properties ◽  
Practical Method ◽  
Living Cells ◽  
Egfr Signaling ◽  
Single Molecule Tracking ◽  
Ras Activation ◽  
And Chemical Properties

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Monofunctional Stealth Nanoparticle for Unbiased Single Molecule Tracking Inside Living Cells

Nano Letters ◽  
2014 ◽  
Vol 14 (4) ◽  
pp. 2189-2195 ◽  
Author(s):  
Domenik Liße ◽  
Christian P. Richter ◽  
Christoph Drees ◽  
Oliver Birkholz ◽  
Changjiang You ◽  
...  
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Tracking

Three-dimensional single-molecule tracking in living cells

2019 ◽  
pp. 229-267
Author(s):  
Daniel M. Kalb ◽  
Duncan P. Ryan ◽  
Demosthenes P. Morales ◽  
Peter M. Goodwin ◽  
James H. Werner
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Living Cells ◽  
Single Molecule Tracking

scholarly journals In Vivo Measurements of Protein Synthesis Kinetics using Single-Molecule Tracking of E.Coli tRNAS

2016 ◽  
Vol 110 (3) ◽  
pp. 351a
Author(s):  
Ivan Volkov ◽  
Javier Aguirre ◽  
Martin Lindén ◽  
Johan Elf ◽  
Magnus Johansson
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Single Molecule Tracking ◽  
In Vivo Measurements
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