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
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
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
