Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells

Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches t...

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Bibliographic Details
Published in:Experimental cell research 2021-07, Vol.404 (1), p.112619-112619, Article 112619
Main Authors: Saito, Takumi, Matsunaga, Daiki, Matsui, Tsubasa S., Noi, Kentaro, Deguchi, Shinji
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ACTIN
Actin-binding proteins
BIOLOGICAL STRESS
Equilibrium constant
FIBERS
FLUORESCENCE
Fluorescence recovery after photobleaching
MOLECULES
MUTANTS
Reaction-diffusion system
Stress fibers
Transgelin-2
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Summary:Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.
ISSN:0014-4827
1090-2422
DOI:10.1016/j.yexcr.2021.112619