In situ study of RSK2 kinase activity in a single living cell by combining single molecule spectroscopy with activity-based probes

The Analyst ◽  
2019 ◽  
Vol 144 (12) ◽  
pp. 3756-3764
Author(s):  
Shengrong Yu ◽  
Zhixue Du ◽  
Chaoqing Dong ◽  
Jicun Ren
Keyword(s):  
Protein Kinases ◽  
Single Molecule ◽  
Living Cell ◽  
Kinase Activity ◽  
Living Cells ◽  
Single Molecule Spectroscopy ◽  
In Situ Study ◽  
Endogenous Protein ◽  
Single Living

FCS with the ABP strategy is a very promising method for studying endogenous protein kinases 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.

In situ investigations of single living cells infected by viruses

1992 ◽  
Vol 42-44 ◽  
pp. 1161-1167 ◽  
Author(s):  
W. Häberle ◽  
J.K.H. Hörber ◽  
F. Ohnesorge ◽  
D.P.E. Smith ◽  
G. Binnig
Keyword(s):  
Living Cells ◽  
Single Living Cells ◽  
Single Living

Adhesion Studies of Single Living Cells Using MEMS Sensors

2000 ◽  
Author(s):  
Chad Sager ◽  
Taher Saif ◽  
Phil LeDuc
Keyword(s):  
Endothelial Cell ◽  
Living Cell ◽  
Mems Sensors ◽  
Adhesion Properties ◽  
Mems Sensor ◽  
Bovine Endothelial Cell ◽  
Single Living Cell ◽  
Single Living Cells ◽  
Single Living

Abstract Adhesion between cells is related to several physiological phenomena such as heart failure (Karila00), how cancer spreads (Ruoslahti99) and if an infection will be fought off. Controlling of these events requires knowledge of how cells adhere. Many previous studies have been conducted with various amounts of success. But none of these methods are independently capable of understanding the adhesion properties of a single living cell. In this article a MEMS sensor has been employed to study, quantitatively and qualitatively, the adhesion properties of a single living bovine endothelial cell. This experiment shows that the strength of a single anchorage site of the endothelial cell to an extracellular matrix coated substrate is 36 nN. Anchorage sites have been observed, in-situ, to be spaced on the order of 1 μm intervals. A model is also proposed for the detachment of a single living cell from a substrate.

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