Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

Abstract

One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host.

Keywords: Candida albicans; Start control; cell size; haploinsufficiency.

Figure 1

The Cln3-Cdc28 kinase complex and the AGC kinase Sch9 control Start in C. albicans. (A) Size distributions of the WT strain (CAI4) as compared to lge mutants cln3/CLN3 and cdc28/CDC28, as well as the whi mutant sch9/SCH9. (B and C) Start is delayed in cln3/CLN3 and cdc28/CDC28 and accelerated in sch9/SCH9. (B) Elutriated G1 phase daughter cells were released into fresh media and monitored for bud emergence as a function of size. (C) G1/S transcription. RNR1 transcript level was assessed by quantitative real-time PCR and normalized to ACT1 levels.

Figure 2

Systematic cell size screen using molecular barcode elutriation and Bar-seq. (A) Centrifugal elutriation separates cells on the basis of size. Progressive increase of the rate of flow of liquid medium counter to the direction of centrifugal force elutes yeast cells of increasingly larger size from the chamber. (B) Size distributions of pooled DBC mutants before (pre-elutriated) and after elutriation of two small cell fractions (28 and 34 ml/min). (C and D) GO terms enrichment of whi (C) and lge (D) mutants (P > 1e−05). GO analysis was performed using GOTermFinder (http://go.princeton.edu/cgi-bin/GOTermFinder). (E) Overlap between C. albicans genes haploinsufficient for cell size and those affecting virulence phenotypes. Avirulent mutant phenotypes were obtained from CGD based on decreased competitive fitness in mice and/or reduced invasion and damage to host cells.