Ras1-induced hyphal development in Candida albicans requires the formin Bni1

Abstract

Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenkörper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.

FIG. 1.

Growth defects of C. albicans formin mutants during yeast growth. Growth of the wild type and formin mutant strains was monitored using time-lapse microscopy over several hours (timescale is in hours:minutes) (A-C). In the movie of the wild type at http://pinguin.biologie.uni-jena.de/phytopathologie/pathogenepilze/index.html, a characteristic change of cell axes after cytokinesis can be observed (previous mother-bud axes are indicated as white, dotted lines and newly established axes as black, dashed lines in panels A to C). This results in lateral movement of cells such that wild-type colonies form a single cell layer (A). In the bni1 mutant, growth was irregular and cells were dispatched in three dimensions. A shift of mother-bud axes occurs but often only due to mechanical forces generated by new buds. The arrows denote enlarged septal sites (B). In the bnr1 mutant, growth axes are kept over several cell cycles, resulting in a linear array of mother and daughter cells (C). Cell shape and budding pattern of the indicated strains were analyzed after staining the cells with calcofluor (D). wt, wild type. Scale bar, 5 μm.

FIG. 2.

Analysis of endocytosis. Endocytosis and the vacuolar morphology of the indicated strains were analyzed by monitoring the uptake of FM4-64 during the yeast stage (A) and hyphal stage (B). In panel A, images of yeast cells that were grown overnight and subsequently stained with FM4-64 are shown. The wild type (wt) and formin mutants generate a single large vacuole. In contrast, the wal1 cells exhibit fragmented vacuoles. Movies 4 to 6 at the website http://pinguin.biologie.uni-jena.de/phytopathologie/pathogenepilze/index.html show the time course of uptake of FM4-64. In panel B, images of FM4-64-stained cells induced to form germ tubes (3 h at 37°C in the presence of serum) are shown. Wild-type and formin mutant germ tubes show the characteristic larger vacuoles in their terminal regions, while the wal1 mutant displays fragmented vacuoles throughout the initial germ tube. Scale bars, 5 μm (A) and 10 μm (B).

FIG. 3.

Time-lapse analysis of the hyphal induction of formin mutants. In vivo time-lapse microscopy was used to monitor the initial phases of germ tube induction in the wild-type (wt), the bni1, and the bnr1 strains on solid media containing serum at 37°C (for complete movies, see movies M7 to -9 at http://pinguin.biologie.uni-jena.de/phytopathologie/pathogenepilze/index.html). Images from these movies acquired at the indicated time points are shown.

FIG. 4.

Mycelial growth defects of the bni1 mutant. The indicated strains were grown for 4 days at 37°C on plates containing either 10% serum or Spider medium prior to photography. Representative images of the edges of colony sectors which demonstrate the mycelial growth defects of the bni1 mutant are shown. wt, wild type.

FIG. 5.

Analysis of the actin cytoskeleton. Images of rhodamine-phalloidin-stained cells of the indicated strains are shown. Cells were grown overnight in yeast extract-peptone-dextrose (YPD) at 30°C, inoculated into fresh YPD (A) or YPD plus 10% serum (B), and grown for 3 hours at 30°C (A) or 37°C (B) prior to fixation and staining. Bar, 10 μm.

FIG. 6.

Localization of Bni1-GFP and the Spitzenkörper. (A) A BNI1-GFP-expressing strain (BNI1/BNI1-GFP) was induced with serum and stained with FM4-64. GFP fluorescence and differential inference contrast (DIC) images were used in an overlay showing the colocalization of Bni1-GFP with the FM4-64-stained Spitzenkörper. (B) Simultaneous localization of Bni1-GFP to the hyphal tip and to a future septal site. Note that in the DIC image no septum is apparent. (C) Comparison of Spitzenkörper morphologies in the wild-type (wt) and bni1 strains using FM4-64. Images of FM4-64-stained germ tubes that were induced by serum at 37°C for 3 hours are shown. Bars, 5 μm.

FIG. 7.

Germ tube induction in C. albicans strains harboring the ras1^G13V allele. Overnight cultures of the indicated strains were grown in liquid medium containing maltose as the sole carbon source to allow for MAL2 promoter-driven expression of the ras1^G13V allele when applicable. Strains were then induced for 3 h with either 10% serum (A) or 10 mM cAMP (B) at 37°C and stained with calcofluor prior to photography. The insets of panel A depict microscopic images of colony edges of the strains grown on solid-medium plates containing serum. Expression of the constitutive ras1^G13V allele did not enable mycelial growth in the bni1 or wal1 mutant strains. wt, wild type.

FIG. 8.

The actin cytoskeleton as a downstream part of signal transduction in regulating polarized morphogenesis. A model is presented in which the central pathways to the actin cytoskeleton regulating endocytosis and secretion are incorporated via Cdc42 into the morphogenetic cascade of C. albicans. Ras1-induced signaling activates the cAMP pathway and a MAP kinase cascade that activates hypha-specific gene expression and may also lead to activation of the Cdc42 module. Defects in the actin cytoskeleton machinery affecting either endocytosis (e.g., via Wal1) or polarisome function (e.g., via Bni1 or Spa2) inhibit mycelial development, indicating the relevance of membranous vesicle transport and cycling for polarized growth. The dashed arrow connecting Cdc42 and Wal1 is to indicate the fact that Wal1 lacks a G protein binding domain and can thus be activated only indirectly by Cdc42. Some other pathways regulating gene expression upon hyphal induction, e.g., the Tup1/Nrg1/Rbf1 pathway and the pH-Rim101 pathway, have been omitted for clarity.