Dual-colour fluorescence microscopy using yEmCherry-/GFP-tagging of eisosome components Pil1 and Lsp1 in Candida...

PCR-based gene targeting technologies have previously been developed for Candida albicans molecular genetic manipulation. Modular marker plasmids for the functional analysis of C. albicans genes have been generated to delete genes, exchange promoters and tag genes with GFP. Here, we have embedded two fluorescent proteins encoded by Venus and yEmCherry into the pFA-plasmid series and demonstrate their usefulness in dual colour microscopy. To this end we analysed the localization of C. albicans homologues of Pil1 and Lsp1, which in S. cerevisiae are components of eisosomes. We find that Pil1/Lsp1-containing eisosomes are cortical protein complexes in C. albicans.Pil1 and Lsp1, tagged with either GFP or yEmCherry, strictly co-localized during all growth stages. Eisosomes, however, localized at distinct positions not overlapping with either cortical actin patches or the endocytosis marker protein Abp1 in yeast or the Spitzenkörper in hyphal cells. To demonstrate the use of Venus yellow fluorescent protein we performed time lapse microscopy of yeast and hyphal stages using a histone H4-Venus tag. As demonstrated, these additions to the toolbox enable a wide range of in vivo applications in C. albicans.