New cell size phenome in C. albicans
We have previously screened 279 C. albicans mutants for their size distribution and uncovered novel potent regulators that modulate size at Start (Chaillot et al. 2019, 2022; Sellam et al. 2019). Here, we expanded our cell size genetic survey to include 1301 non-essential mutants from the GRACE (Gene Replacement and Conditional Expression) collection, which bear a gene deletion at one locus and an integrated tetracycline-regulated allele at the other locus (Roemer et al. 2003).
non-essential mutants. A total of 18 mutants were in common with the set of mutants investigated in our former work (Sellam et al. 2019), which leave the opportunity to analyse 1283 new mutants for cell size defect (representing ~ 30% of all predicted non-essential genes in C. albicans).
Clustering of size distributions of the GRACE collection discriminated both large and small mutants (which we refer as lge and whi phenotypes, respectively) from the rest of the conditional mutants with comparable size to the parental wild-type (WT) strain CAI4 (Fig. 1A and Table S1). We defined a significant change in size as a 20% increase or decrease in median size, as compared to WT, as applied in our previous screen (Sellam et al. 2019). Accordingly, a total of 40 whi and 11 lge mutants were identified (Table S2). Size phenotypes of two repressible mutants, including the swi6 and ssk1 previously shown as large and small mutants (Sellam et al. 2019), respectively, were confirmed by our analysis (Fig. 1B).
Gene Ontology (GO) enrichment analysis revealed that small size mutants were predominantly defective in functions related to rRNA processing and ribosome biogenesis (Fig. 1C and 1F). A similar finding was reported in S. cerevisiae and also in our former surveys of size phenome in C. albicans (Jorgensen et al. 2002; Soifer and Barkai 2014; Chaillot et al. 2017; Sellam et al. 2019). As ribosome biogenesis dictates the rate of protein production and thus underlies the cell’s capacity to grow (Jorgensen et al. 2004; Jorgensen and Tyers 2004), the small size of those mutants could be explained by the fact that they passed Start with a reduced cell mass.
Inactivation of many genes with mitochondrial functions including mitochondrial genome maintenance and organization (IRC3, FIS1, MDM20, MSU1) and, protein import and assembly (MJD1, COX19) led to small size mutants (Fig. 1C and 1H). Genes whose repression led to whi phenotype were also enriched in cytokinesis, specifically in the formation of the contractile actomyosin ring (MYO1, IQG1, NAT3) (Fig. 1C and 1E). Disruption of many other genes also reduced cell size in C. albicans, including genes implicated in amino acid metabolism (SSY1, CAR2, ARO80, OPT7), carbon metabolism (ICL1, orf19.1797, RTG1), amongst many other processes (Fig. 1J and Table S2). Conversely, mutations in processes related to cell cycle regulation (swi6, lte1), chromatin control (hst1, zds1) and cell wall organization (kre1, chs3) conferred an increase of cell size (Fig. 1D and 1I-J). Overall, much as in the case of other species, cell size in C. albicans is a complex trait that depends on diverse biological processes.
Limited overlap of the fungal cell size phenome across species
C. albicans and S. cerevisiae represent yeast genera separated by ~ 70M years of evolution (Salichos and Rokas 2013) and share the morphological trait of budding, as well as core cell cycle and growth regulatory mechanisms (Berman 2006; Cote et al. 2009). However, an extensive degree of rewiring of both cis-transcriptional regulatory circuits and signalling pathways was uncovered across many cellular and metabolic processes between the two yeasts (Lavoie et al. 2009; Blankenship et al. 2010; Li and Johnson 2010). To assess the extent of conservation and plasticity of the size phenome between the two species, genes that affect cell size in C. albicans were compared to their corresponding orthologs in S. cerevisiae. Surprisingly, we found minimal overlap between homozygous size mutants in both species (Fig. 2A). Only one small size mutant was common between C. albicans and S. cerevisiae, namely zds1, a regulator of both mitotic entry and exit (Rossio and Yoshida 2011). Similarly, only one large size mutant was shared, that is that of the SBF subunit Swi6, a transcriptional regulator of the G1/S transition (Jorgensen and Tyers 2004). Unexpectedly, the C. albicans small sized mutant mdm20 exhibited a large phenotype in S. cerevisiae. To confirm these disparities in the size control network independently of the particular cut-offs used to define large and small mutants in different studies, we examined the relationship between median size of all screened C. albicans GRACE mutants and their counterparts in S. cerevisiae. Overall, no correlation was observed between the size phenomes of the two species (R2 = 0.0146; Fig. 2B).
Modulation of Start by novel and known mechanisms
Previous work has shown that mutations affecting core cellular process such as ribosomal and nucleolar proteins and cell division lead to a dramatically decreased growth rate and corresponding size defect (Tyson et al. 1979; Jorgensen et al. 2002). To identify bona fide Start regulators, rather than slow growth mutants, mutations that significantly impacted growth rate were excluded from our dataset. We calculated the doubling times of the 51 size mutants identified in this work and those that had more than 10% increase in doubling time as compared to the parental WT strains were omitted. A short list of 33 ( 26 whi and 7 lge mutants) strains was retained as high confidence size mutants of C. albicans (Table 1). Start onset of five potent C. albicans Start regulators Myo1, Iqg1, Chs3, Kre1 and Sun41 in addition to the conserved regulators Swi6, were characterized by assessing bud emergence in G1-elutrated cells of their mutant strains. As shown in Fig. 2C, the critical cell size was significantly reduced in myo1 and iqg1 suggesting an accelerated Start in these whi mutants. Conversely, chs3, kre1, sun41 and swi6 Lge mutants passed Start at larger size as compared to the WT. These data support that the processes related to cell wall biogenesis and actin ring constriction are critical determinant of Start onset and cell size homeostasis. The larger size of cell wall mutants could be explained by a prolonged G1 phase as a consequence of a reduced amount of newly synthesised cell wall materials required for the formation of the daughter cell and the septal wall.
Table 1
Potent Start regulators in C. albicans.
Size gene | Orf19 ID | Median size (fL) | Size reduction or increase /WT(%) | Doubling time (min) | Function | Homolog in S. cerevisiae |
Small size mutants |
MYO1 | orf19.6294 | 42.3 | 38.1 | 100.56 | component of actomyosin ring | YHR023W |
- | orf19.2064 | 50.5 | 26.1 | 106.25 | transcription factor with zinc finger DNA-binding motif | - |
DET1 | orf19.6747 | 51.2 | 25.0 | 111.25 | phosphatase of unknown function | YDR051C |
COX19 | orf19.4967 | 53.9 | 21.1 | 113.75 | cytochrome c oxidase assembly protein | YLL018CA |
- | orf19.6681 | 49.8 | 27.1 | 115.05 | protein of unknown function | - |
IQG1 | orf19.6536 | 45.7 | 33.1 | 122.5 | protein that enhances actin ring formation | YPL242C |
STB5 | orf19.3308 | 53.9 | 21.1 | 123.03 | transcription factor with zinc cluster DNA-binding motif | YHR178W |
CAR2 | orf19.5641 | 53.9 | 21.1 | 125.58 | L-ornithine transaminase | YLR438W |
NAA25 | orf19.6071 | 53.3 | 22.0 | 126.25 | non-catalytic subunit of the NatB N-terminal acetyltransferase | YOL076W |
- | orf19.7010 | 53.9 | 21.1 | 129.01 | protein of unknown function | YIL001W |
ARO80 | orf19.3012 | 51.2 | 25.0 | 130.3 | transcriptional activator of aromatic amino acid catabolism | YDR421W |
CWC23 | orf19.3785 | 53.9 | 21.1 | 131.25 | protein involved in pre-mRNA splicing | YGL128C |
- | orf19.2106 | 53.3 | 22.0 | 131.53 | protein of unknown function | YLR326W |
FIS1 | orf19.7111 | 51.9 | 24.0 | 134.31 | protein involved in mitochondrial fission | YIL065C |
IRC3 | orf19.2798 | 53.9 | 21.1 | 136.4 | putative helicase | YDR332W |
| orf19.7336 | 53.9 | 21.1 | 136.98 | predicted membrane transporter | - |
UBP1 | orf19.7367 | 53.9 | 21.1 | 137.26 | ubiquitin-specific protease | YDL122W |
MDJ2 | orf19.3574 | 52.6 | 23.0 | 137.83 | constituent of the mitochondrial import motor | YNL328C |
YIP5 | orf19.4922 | 51.2 | 25.0 | 137.86 | protein that interacts with Rab GTPases | YGL161C |
MSU1 | orf19.3624 | 51.9 | 24.0 | 137.98 | component of the mitochondrial degradosome | YMR287C |
SSK1 | orf19.5031 | 52.6 | 23.0 | 138.8 | response regulator of two-component system | YLR006C |
ERV29 | orf19.4579 | 51.2 | 25.0 | 139.6 | protein involved in vesicle formation | YGR284C |
RQT4 | orf19.2391 | 51.9 | 24.0 | 140.48 | subunit of ribosome-associated quality control trigger complex (RQT) | YKR023W |
- | orf19.2996 | 50.5 | 26.1 | 140.9 | ORF, Uncharacterized | YLR436C |
TIP41 | orf19.3937 | 53.9 | 21.1 | 146.73 | protein of TOR signaling pathway | YPR040W |
RTG1 | orf19.4722 | 53.9 | 21.1 | 149.63 | transcriptional regulator of galactose utilization | YOL067C |
Large size mutants |
HST1 | orf19.4761 | 88.1 | 29.0 | 140.4 | histone deacetylase | YOL068C |
CHS3 | orf19.4937 | 81.9 | 19.9 | 141.1 | chitin synthase | YBR023C |
KRE1 | orf19.4377 | 90.1 | 31.9 | 142.1 | protein involved in beta-glucan assembly | YNL322C |
PEX14 | orf19.1805 | 82.6 | 20.9 | 143.1 | component of the peroxisomal importomer complex | YGL153W |
SUN41 | orf19.3642 | 93.5 | 36.9 | 146.1 | cell wall glycosidase | YNL066W |
TRM1 | orf19.3265 | 86.7 | 26.9 | 148.1 | tRNA methyltransferase | YDR120C |
SWI6 | orf19.4725 | 100.4 | 47.0 | 151.1 | component of the MBF and SBF transcription complexes involved in G1/S cell-cycle progression | YLR182W |
WT size |
CAI4 | - | 68.3 | - | 138 | - | - |
Constriction of the actomyosin ring formed by F-actin and the type II myosin Myo1 is essential for cytokinesis and the completion of the mitotic cell cycle (Cheffings et al. 2016). In the budding yeast, the actomyosin contractile ring is coordinated with and guides the formation of the primary septum, which deposit new cell wall materials between the dividing cells (Juanes and Piatti 2016). As many components of the C. albicans actomyosin ring act as potent size regulators, it is tempting to speculate that cell size is also monitored at the G2/M transition prior to cytokinesis. Alternatively, this suggests that in addition to the Start checkpoint of the mother cell, size could be gauged sequentially at G2/M by ensuring that the daughter cell have reached an optimal size to promote cytokinesis. A similar mechanism of size control at G2/M was proposed in S. cerevisiae whereby Swe1, a protein kinase that coordinates entry into mitosis at the G2/M transition, was shown to activate a cell size checkpoint to monitor cell size of the daughter cell (Harvey and Kellogg 2003; Turner et al. 2012).
Inactivation of genes related to ribosome biogenesis and carbon and nitrogen metabolism resulted in altered cell size (Fig. 1C and 1G, Table 1), consistent with our previous finding underscoring the role of these processes in the timing of Start onset and critical cell size (Chaillot et al. 2019, 2022; Sellam et al. 2019). Perturbation of mitochondrial genes led to a whi phenotype which might be linked to a decrease of energy supply or metabolic capacity of the cells (Fig. 1C). In metazoan, growth factor signaling such as the insulin/mTOR (mammalian target of rapamycin) pathway is essential for size homeostasis, a process that requires the contribution of mitochondria (Lloyd 2013; Miettinen and Björklund 2016). Future efforts describing the specific mitochondrial process required for size control in C. albicans will lend further insights into how fungal mitochondria signal to the Start machinery.