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.

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 Dynamic recruitment of phospholipase Cγ at transiently immobilized GPI-anchored receptor clusters induces IP3–Ca2+ signaling: single-molecule tracking study 2

2007 ◽  
Vol 177 (4) ◽  
pp. 731-742 ◽  
Author(s):  
Kenichi G.N. Suzuki ◽  
Takahiro K. Fujiwara ◽  
Michael Edidin ◽  
Akihiro Kusumi
Keyword(s):  
Single Molecule ◽  
Lateral Diffusion ◽  
Single Molecule Tracking ◽  
Receptor Clusters

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Gαi2 and Lyn via protein–protein and raft interactions. Lyn is activated probably by the Gαi2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLCγ2 and CD59 clusters revealed that PLCγ2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP3–Ca2+ signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP3–Ca2+ signal, via PLCγ2 recruitment. The prolonged, analogue, bulk IP3–Ca2+ signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP3 bursts, each created by the transient recruitment of PLCγ2 molecules to STALLed CD59.

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 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 Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation

2020 ◽  
Vol 21 (8) ◽  
pp. 2803 ◽  
Author(s):  
Marie-Lena I.E. Harwardt ◽  
Mark S. Schröder ◽  
Yunqing Li ◽  
Sebastian Malkusch ◽  
Petra Freund ◽  
...  
Keyword(s):  
Single Molecule ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Super Resolution ◽  
Cell Types ◽  
Surface Expression ◽  
Living Cells ◽  
Ligand Activation ◽  
Receptor Clusters ◽  
Super Resolution Microscopy

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

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
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