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

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.

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 Direct measurements of mRNA translation kinetics in living cells

2020 ◽  
Author(s):  
Mikhail Metelev ◽  
Ivan L. Volkov ◽  
Erik Lundin ◽  
Arvid H. Gynnå ◽  
Johan Elf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mrna Translation ◽  
Living Cells ◽  
Single Molecule Tracking ◽  
Ribosomal Subunits ◽  
Reading Frame ◽  
Direct Measurements ◽  
Translation Start ◽  
Labeling Method ◽  
Correct Translation

ABSTRACTRibosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.

scholarly journals TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shipeng Shao ◽  
Hongchen Zhang ◽  
Yong Zeng ◽  
Yongliang Li ◽  
Chaoying Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Time Course ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Spatiotemporal Resolution ◽  
Dynamic Interactions ◽  
Single Molecule Tracking

AbstractProtein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

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