Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Abstract

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 ± 0.30 μm/sec in R256H cells compared to 1.60 ± 0.42 μm/sec in wild type cells, p < 0.005). Video LinkThe video component of this article can be found at

G65V Substitution in Actin Disturbs Polymerization Leading to Inhibited Cell Elongation in Cotton

“…Over the past few decades, hundreds of actin mutants, responsible for various morphological changes, have been identified in both uni- and multi-cellular organisms (Ishiguro and Kobayashi, 1996; Feng and Marston, 2009; Kato et al, 2010). Actin mutants in human are the most thoroughly studied because they are causal for genetic diseases (Pierick et al, 2014; Marston, 2018). At the molecular level, these mutated actins were unable to fold properly to form functional actin, were incapable of polymerization, or had changes in their ability to interact with ABPs (Costa et al, 2004; Feng and Marston, 2009).…”