A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi

Here we report the construction and application of a range of expression plasmids designed to facilitate livecell imaging of F-actin dynamics in filamentous fungi simultaneously with other, preferably GFP-tagged fusion proteins. Pros and cons of the use of three different red fluorescent proteins (RFPs), two different promoters and three different selection markers are addressed. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol57/iss1/4 8 Fungal Genetics Reports A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi Alexander Lichius and Nick D. Read Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JR, UK; Alex@fungalcell.org Fungal Genetics Reports 57:8-14 Here we report the construction and application of a range of expression plasmids designed to facilitate live-cell imaging of F-actin dynamics in filamentous fungi simultaneously with other, preferably GFP-tagged fusion proteins. Pros and cons of the use of three different red fluorescent proteins (RFPs), two different promoters and three different selection markers are addressed. Live-cell imaging of F-actin dynamics is possible in a wide range of eukaryotes. Lifeact is a 17 aa peptide derived from the actin-binding protein 140 (Abp140) of S. cerevisiae which specifically binds to filamentous actin (F-actin) (Riedl et al., 2008). Functionality of green fluorescent Lifeact reporters (Lifeact-GFP) for the visualisation of F-actin structures in living cells has so far been documented for three of the four eukaryotic phyla, including yeasts and filamentous fungi (Berepiki et al., 2010; Böhmer et al., 2009; Coffman et al., 2009; Delgado-Álvarez et al., 2010; Riedl et al., 2008), plants (Era et al., 2009; Smertenko et al., 2010; Vidali et al., 2009), and mammals (Estecha et al., 2009; Riedl et al., 2008; Riedl et al., 2010). The application of Lifeact-GFP in filamentous fungi has been pioneered in the ascomycete Neurospora crassa (Berepiki et al., 2010; Delgado-Álvarez et al., 2010), and is currently being adopted for the use in numerous other fungal species. In basidiomycetes, however, labelling properties of Lifeact appear to be restricted to the visualization of septal rings, as F-actin cables and patches have so far not been explicitly reported (Böhmer et al., 2009). Lifeact-GFP reporters work equally well in fungi that use the CUG codon for serine and not leucine, such as Candida sp. (Kawaguchi et al., 1989), once they have been codon corrected, (E. Epp, McGill Univ. Montreal, pers.comm.). Why RFP and which one? The most widely used fluorescent reporter for live-cell imaging analyses of protein dynamics in filamentous fungi is GFP. To allow simultaneous observation of F-actin with any other GFP-tagged protein, the development of a Lifeact probe (Riedl et al., 2008) labelled with a different color was desired. As green and red emission signals can be spectrally well separated, and fluorescence microscopes with the corresponding excitation and emission detection settings are widely available, we aimed to generate a functional Lifeact-RFP reporter construct. The first red fluorescent Lifeact probe that we adopted for the filamentous model fungus Neurospora crassa was Lifeact-tdTomato (Roca et al., 2010). Although Lifeact-tdTomato reliably labelled F-actin patches, cables and septal rings (Figure 1), this reporter occasionally produced additional ring-shaped structures (~1-2 μm in diameter, Figures 1B and C), which we suggest may be formed through spontaneous self-assembly of F-actin, destabilized through the interaction with the Lifeact-tdTomato construct, and native septins. Evidence for this comes from previous studies indicating that due to sterical hindrance, mRFP and tdTomato fusion constructs failed to correctly localize microtubules and connexin (Shaner et al., 2004; Shaner et al., 2008). This not well understood property of some RFPs might have potentiated mislocalization artefacts of the overexpressed Lifeact-tdTomato fusion construct leading to the formation of these cytoplasmic rings from destabilized F-actin. Interestingly, similar structures have been reported to form upon Latrunculin A treatment in mammalian cells (Kinoshita et al., 2002) and haploid cells of Ustilago maydis (Böhmer et al., 2009). Appearance of these rings in N. crassa was resolved by exchanging tdTomato for the monomeric red fluorescent protein TagRFP (Shaner et al., 2008). Figure 1. Lifeact-RFPs labelled F-actin cables, patches and rings. (A) Two fused wt cells expressing LifeactTagRFP. F-actin cables stretched throughout the spore body of the upper cell, whilst actin patches were distributed over the surface of the bottom cell. Dense arrays of actin cables were localized at the cell fusion site (arrowhead), and condensed into an actin ring prior to septum formation (arrow). (B to D) Examples of wt conidia expressing Lifeact-tdTomato. (B) Conidial germling with actin cables aligned with the long axis of the germ tube. An extremely bright fluorescent ring (arrow) in the cytoplasm is obvious but not associated with any known cellular structure. Similar rings have been described in other model systems and are likely the product of spontaneous self-assembly of the Lifeact-tdTomato construct with native septins (see text for details). Also vacuolar accumulation of the fusion construct created minor artefactual background fluorescence. (C) Two fusing germlings with bright fluorescent rings at different localizations within the cells. (D) Conidia in the process of CAT homing (Read et al., 2009), showing intense accumulation of actin patches and cables in the tip of the smaller germling (arrowhead). Formation of a cortical actin ring at the spore neck is again indicated with an arrow. All images show maximum intensity projection of deconvolved z-stacks. Scale bars, 5 μm. Published by New Prairie Press, 2017