Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.DOI: http://dx.doi.org/10.7554/eLife.24903.001

SummaryWe investigated the myosin expression profile in prostate cancer cell lines and found that Myo1b, Myo9b, Myo10, and Myo18a were expressed at higher levels in cells with high metastatic potential. Moreover, Myo1b and Myo10 were expressed at higher levels in metastatic tumors. Using an siRNA-based approach, we found that knockdown of each myosin resulted in distinct phenotypes. Myo10 knockdown ablated filopodia and decreased 2D migration speed. Myo18a knockdown increased circumferential non-muscle myosin 2A-associated actin filament arrays in the lamella and reduced directional persistence of 2D migration. Myo9b knockdown increased stress fiber formation, decreased 2D migration speed, and increased directional persistence. Conversely, Myo1b knockdown increased numbers of stress fibers but did not affect 2D migration. In all cases, the cell spread area was increased and 3D migration potential was decreased. Therefore, myosins not only act as molecular motors but also directly influence actin organization and cell morphology, which can contribute to the metastatic phenotype.

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.

Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145–3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

Antibodies have been indispensable tools in molecular biology, biochemistry and medical research. However, a number of issues surrounding validation, specificity and batch variation of commercially available antibodies have prompted research groups to develop novel non-antibody binding reagents. The ability to select highly specific monoclonal non-antibody binding proteins without the need for animals, the ease of production and the ability to site-directly label has enabled a wide variety of applications to be tested, including imaging. In this review, we discuss the success of a number of non-antibody reagents in imaging applications, including the recently reported Affimer.