F-Actin Dynamics in Neurospora crassa

Abstract

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p.

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

“…We also found that Pccg-1 did not allow satisfactory visualization of Lifeact-FPs in mature vegetative hyphae (Berepiki et al, 2010). To fully overcome these problems and guarantee stable fluorescent signal levels throughout the whole life cycle of the fungus, the promoter of the translation elongation factor-1 (locus NCU02003.4) (Ptef-1) of Neurospora crassa was alternatively employed (Berepiki et al, 2010). In summary, for studies during the first 6-7 h of conidial development, Pccg-1 is a very well suited promoter for N. crassa, whereas expression under its control appears less consistent during the transition from the germling phase into mature hyphal growth.…”

“…Furthermore, TagRFP-T, in contrast to TagRFP, although showing a small, ~17% decrease in brightness, shows virtually no emission in the green part of the spectrum (Shaner et al, 2008). Therefore, Lifeact-TagRFP-T is ideally suited for co-imaging with GFP-tagged proteins, for example BML-sGFP which reliably labels β-tubulin in microtubules (Berepiki et al, 2010;Freitag et al, 2004).…”

“…In conidial germlings we consistently observed a noticeable decrease in expression and thus recordable signal intensity after 6-7 h of incubation (about 5-6 h after cell symmetry breaking of the germinating conidium). We also found that Pccg-1 did not allow satisfactory visualization of Lifeact-FPs in mature vegetative hyphae (Berepiki et al, 2010). To fully overcome these problems and guarantee stable fluorescent signal levels throughout the whole life cycle of the fungus, the promoter of the translation elongation factor-1 (locus NCU02003.4) (Ptef-1) of Neurospora crassa was alternatively employed (Berepiki et al, 2010).…”

“…Scale bars, 5 µm. To ensure an equivalent high level of fluorescence regardless of its use as a N-or C-terminal fusion tag the original TagRFP was modified through the addition of GFP-like N-and C-terminal residues (VSKGEE and LNGMDELYK, respectively), thereby improving its utility for 4D live-cell imaging (Berepiki et al, 2010;Shaner et al, 2004;Shaner et al, 2008). Finally, the photostability of this optimized TagRFP version was increased through site-directed mutagenesis (SDM) of S162T, using the oligonucleotides TagRFP_S162T_fw 5'-CCTGGAAGGCAGAACCGACATGGCCCTGAAGC-3' and its reverse complement TagRFP_S162T_rv 5'-GCTTCAGGGCCATGTCGGTTCTGCCTTCCAGG-3' (the mismatching nucleotide conferring S162T exchange is underlined; the affected triplet is in bold and italicised).…”

“…pAL3-Lifeact was assembled using InFusion PCR cloning (http://www.clontech.com) to recombine the insert encoding for LifeactTagRFP amplified from pJT603f (Jens Tilsner, University of Edinburgh) with the EcoRV/BamHI linarized pBARGRG-1 in a ligationindependent step (Berepiki et al, 2010). Exchange of the ignite resistance cassette (PtrpC::bar) for the nourseothricin resistance cassette (PtrpC::nat1) was accomplished using the same technique to recombine the PtrpC::nat1 insert amplified from pG-Nat1 (Kück and Hoff, 2006) with SpeI/XbaI linearized pAL3-Lifeact, resulting in pAL4-Lifeact.…”