Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells

Anna Lopata,Michelle Peckham,Christian Tiede,Peter J Knight,Sarah M Heissler,Ruth Hughes,James R Sellers,Darren Tomlinson

doi:10.1038/s41598-018-24953-4

Abstract

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Highlights

The actin cytoskeleton is widely visualised in cell biology, owing to the role it plays in cellular processes, including intracellular transport, migration, cell shape and gene regulation
It has been reported to enable a clearer visualisation of the actin cytoskeleton than eGFP-actin[17], it does not recognize all actin structures in cells
The binding of the purified Affimers to F-actin in vitro was tested using a sedimentation assay taking advantage of the ability for actin filaments to be pelleted in the ultracentrifuge at 110,000 g (Fig. 2A)

Summary

Introduction

The actin cytoskeleton is widely visualised in cell biology, owing to the role it plays in cellular processes, including intracellular transport, migration, cell shape and gene regulation. Visualizing actin by expressing eGFP-actin has been used for some time[12] but has numerous drawbacks As both G-actin and F-actin are labelled, there is a higher fluorescent background from non-filamentous actin[13], and expression of eGFP-actin[14] can affect cell behaviour[15] and see a recent review[1]. This has led to the development of several fluorescent F-actin-binding peptides (e.g. LifeAct and F-tractin) to avoid this problem. Lifeact consists of eGFP fused to the C-terminus of the first 17 amino acids from the yeast actin-binding protein Abp14016. We demonstrate the potential ability to further isolate Affimers capable of recognizing different forms of actin

Methods

Results

Conclusion