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 GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

2007 ◽  
Vol 177 (4) ◽  
pp. 717-730 ◽  
Author(s):  
Kenichi G.N. Suzuki ◽  
Takahiro K. Fujiwara ◽  
Fumiyuki Sanematsu ◽  
Ryota Iino ◽  
Michael Edidin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Lipid Raft ◽  
Single Molecules ◽  
Lateral Diffusion ◽  
Living Cells ◽  
Cross Linking ◽  
Single Molecule Tracking ◽  
Signaling Mechanisms ◽  
Subsequent Activation ◽  
Receptor Clusters

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold–induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Gαi2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein–protein and lipid–lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP3 production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Gαi2. Because both Gαi2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Gαi2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

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

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

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.

scholarly journals Actin kinetics shapes cortical network structure and mechanics

2016 ◽  
Vol 2 (4) ◽  
pp. e1501337 ◽  
Author(s):  
Marco Fritzsche ◽  
Christoph Erlenkämper ◽  
Emad Moeendarbary ◽  
Guillaume Charras ◽  
Karsten Kruse
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Length Distribution ◽  
Living Cells ◽  
Stochastic Simulations ◽  
Cortical Actin ◽  
Animal Cells ◽  
Cellular Mechanics ◽  
Cellular Processes ◽  
Actin Cortex

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

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
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