The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells

摘要: 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 probe to detect organization and dynamics cytoskeleton living cells. It generally assumed that expression fusion does not disturb distribution endogenous throughout cell, reflects actin. However, we noticed EGFP-β-actin caused excessive formation microfilaments several mammalian cell lines. To investigate whether position EGFP tag on affected filaments, constructed an vector harboring β-actin-EGFP gene. In contrast EGFP-β-actin, cells expressing showed filaments high background from monomer cytosol. Additionally, detergent insoluble assay revealed majority detergent-insoluble was recovered pellet. Furthermore, found affects migration NBT-L2b mechanical stiffness U2OS These results indicate N-terminus tend form filaments. addition, EGFP-actin both morphological physiological phenotypes compared actin-EGFP.Key words: actin, GFP, probe.