RRC ID 54630
Author Nagasaki A, T Kijima S, Yumoto T, Imaizumi M, Yamagishi A, Kim H, Nakamura C, Q P Uyeda T.
Title The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells.
Journal Cell Struct Funct
Abstract Actin, a major component of microfilaments, is involved in various eukaryotic cellular functions. Over the past two decades, actin fused with fluorescent protein has been used as a probe to detect the organization and dynamics of the actin cytoskeleton in living eukaryotic cells. It is generally assumed that the expression of fusion protein of fluorescent protein does not disturb the distribution of endogenous actin throughout the cell, and that the distribution of the fusion protein reflects that of endogenous actin. However, we noticed that EGFP-β-actin caused the excessive formation of microfilaments in several mammalian cell lines. To investigate whether the position of the EGFP tag on actin affected the formation of filaments, we constructed an expression vector harboring a β-actin-EGFP gene. In contrast to EGFP-β-actin, cells expressing β-actin-EGFP showed actin filaments in a high background from the monomer actin in cytosol. Additionally, the detergent insoluble assay revealed that the majority of the detergent-insoluble cytoskeleton from cells expressing EGFP-β-actin was recovered in the pellet. Furthermore, we found that the expression of EGFP-β-actin affects the migration of NBT-L2b cells and the mechanical stiffness of U2OS cells. These results indicate that EGFP fused to the N-terminus of actin tend to form excessive actin filaments. In addition, EGFP-actin affects both the cellular morphological and physiological phenotypes as compared to actin-EGFP.Key words: actin, GFP, cytoskeleton and probe.
Volume 42(2)
Pages 131-140
Published 2017-1-1
DOI 10.1247/csf.17016
PMID 28855440
MeSH Actin Cytoskeleton / chemistry Actin Cytoskeleton / metabolism* Actins / analysis Actins / chemistry Actins / metabolism* Animals Cell Line Cytosol / chemistry Cytosol / metabolism Green Fluorescent Proteins / analysis Green Fluorescent Proteins / metabolism* Humans Mammals
IF 3.5
Times Cited 7
Arabidopsis / Cultured plant cells, genes rpc00008
Human and Animal Cells HeLa 293(RCB1637)
DNA material EGFP-beta-actin (RDB20216) beta-actin-EGFP (RDB20217)