Rac1 and Cdc42 have different roles in Candida albicans development

Abstract

We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen Candida albicans. We identified and disrupted RAC1 and show here that, in contrast to CDC42, it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in C. albicans development.

FIG. 1.

Comparison of Rac1 proteins from different species. C. albicans (Ca), Y. lipolytica (Yl), U. maydis (Um), C. elegans (Ce), D. melanogaster (Dm), and human (Hs) proteins were compared. Gray boxes indicate sequence identity.

FIG. 2.

Characterization of C. albicans RAC1 deletion mutant. (A) RAC1 deletion construction. PCR analysis was performed with total genomic DNAs from wild-type (PY82) and rac1Δ/RAC1 (PY256) and rac1Δ/rac1Δ (PY191) mutant strains, using the primer pair RAC1.P5 and RAC1.P6. For the rac1Δ/rac1Δ mutant strain, no 0.8-kb RAC1 band was detected. (B) RAC1 is not expressed in the deletion mutant. The levels of RAC1 and CDC42 transcripts in the wild-type and rac1Δ/rac1Δ mutant strains were quantified by qRT-PCR, using RAC1.P11 and -P12 or CDC42.P3 and -P4 primers. Values are relative to the actin message, amplified with previously described primers (4). (C) Rac1 is not required for viability. Dilutions of the indicated strains were spotted on YEPD plates and grown for 3 days. (D) Rac1 is not required for actin cytoskeleton organization. Differential interference contrast and deconvoluted fluorescence images of the indicated strains stained with Alexa-phalloidin are shown. Fluorescence images are maximum intensity projections of z sections (11 to 15 by 0.1 μm).

FIG. 3.

C. albicans Rac1 binds GTP. (A) Immunoblot analysis of Cdc42 and Rac1. Extracts of GFP-Cdc42 (PY196)- and GFP-Rac1 (PY205)-expressing cells were analyzed by SDS-PAGE, followed by immunoblotting and probing with anti-GFP polyclonal sera. (B) CRIB pull-down assay with C. albicans Rac1. Clarified cell lysates of cells expressing GFP-Rac1, dominant active GFP-Rac1(G12V) (PY209), dominant negative GFP-Rac1(T17N) (PY212), or no GFP-Rac1 (PY191) were incubated with CRIB-glutathione S-transferase-glutathione-agarose resin. Eluted proteins were separated by SDS-PAGE and analyzed as in panel A. Lanes 5 to 8 were loaded with 1% of total Rac1.

FIG. 4.

Rac1 is essential for embedded medium-induced but not serum-induced filamentous growth. (A) Serum-induced filamentous growth. Wild-type (PY82) and rac1Δ/rac1Δ mutant (PY191) strains were grown in the presence of YEPD and FCS. Images are of colonies grown for 5 days (upper panel) and of cells incubated for 3 h at 37°C in liquid medium (lower panel). (B) Embedded medium-induced filamentous growth. Images of colonies of the wild type, the rac1Δ/rac1Δ mutant strain, the rac1Δ/rac1Δ mutant strain complemented with RAC1 (PY275), and the cdc42Δ/PMETCDC42 mutant strain (PY123) were taken after 6 days of growth at 25°C embedded in agar containing YEP plus sucrose. (C) Percentages of filamentous colonies in the strains illustrated in panel B and the rac1Δ/rac1Δ PADHGFPRAC1 mutant strain (PY205). Averages of four to six experiments (n = 200 colonies each) are shown, with standard deviations indicated.

FIG. 5.

Rac1 and Cdc42 localize to the plasma membrane with different dynamics. (A) Rac1 localizes uniformly to the plasma membrane in yeast and hyphal cells. rac1Δ/rac1Δ mutant cells expressing GFP-Rac1 (PY205) and cdc42Δ/PMETCDC42 mutant cells expressing GFP-Cdc42 (PY196) were used for FRAP analyses. PY196 cells were grown in medium containing 2.5 mM methionine and cysteine to repress the expression of endogenous CDC42. Confocal microscopy images of budding cells or cells undergoing hyphal growth in response to serum at 37°C for 2 h were taken prior to and subsequent to (10 to 100 ms) photobleaching and after fluorescence recovery. The arrowheads indicate the bleached areas. (B) Single-phase exponential curve fits of fluorescence recovery after photobleaching intensities. Average intensities of bleached areas normalized for photobleaching during image acquisition are shown. Arrowheads indicate the times of the images illustrated in panel A.

FIG. 6.

Rac1 and Cdc42 cannot substitute for one another in C. albicans growth. (A) Rac1 overexpression does not complement for viability in cdc42Δ/PMETCDC42 mutant strains. A dilution series of the cdc42Δ/PMETCDC42 mutant strain overexpressing GFP-Cdc42 (PY196) or GFP-Rac1 (PY225) was spotted on plates of the indicated media and incubated for 3 days. (B) Rac1 overexpression does not complement the Cdc42 requirement for serum-induced hyphal growth. Images of exponentially growing cells from the wild-type strain (PY82), the cdc42Δ/PMETCDC42 mutant strain (PY123), and the cdc42Δ/PMETCDC42 mutant strain overexpressing Cdc42 (PY115), GFP-Cdc42, Rac1 (PY317), or GFP-Rac1 were taken after growth in the absence (YEPD alone) or presence of FCS (50% YEPD, 50% FCS) for 3 h.