Protein Detection and Localization in Plant Cells Using Spot-Tagging.

Abstract

Fluorescent labelling of proteins enables the determination of their spatiotemporal localization but, sometimes, it can perturb their activity, native localization, and functionality. Spot-tag is a12-amino acid peptide recognized by a single-domain nanobody and could potentially resolve the issues associated with large fluorescence tags due to its small size. Here, using as an example the microtubule motor CENTROMERIC PROTEIN E-RELATED KINESIN 7.3 (KIN7.3), we introduce the spot-tag for protein labelling in fixed and living plant cells. Spot-tagging and detection by an anti-spot nanobody of ectopically expressed KIN7.3 did not interfere with its native localization. Most importantly, our spot-tagging pipeline facilitated the localization of KIN7.3 much more rapidly and likely accurately than labelling with large fluorescent proteins or even immunolocalization approaches. We should, though, note some limitations we have not resolved yet. Spot-tagging is functional only in fixed cells; it is available only as two fluorophores and may create a noisy background during imaging. However, we foresee that, besides the limitations of this method, spot-tagging will apply to many proteins, offsetting activity perturbations and low photon quantum yields of other protein-tagging approaches.