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1 A phylogenetically-restricted essential cell cycle progression factor in the

2 human pathogen Candida albicans
3

5 Priya Jaitly1, Mélanie Legrand2, Abhijit Das1†, Tejas Patel1†, Murielle Chauvel2, Christophe
6 d’Enfert2* and Kaustuv Sanyal1, 3*
7

8
1
9 Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru
10 Centre for Advanced Scientific Research, Bangalore, India.
2
11 Institut Pasteur, Université de Paris, INRAE, USC2019, Unité Biologie et Pathogénicité
12 Fongiques, F-75015 Paris, France.
3
13 Osaka University, Suita, Osaka, Japan.
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15

16 * Corresponding authors. Email: [Link]@[Link], sanyal@[Link]

17 † These authors contributed equally to this work.
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33 Abstract
34

35 Chromosomal instability in fungal pathogens caused by cell division errors is associated with
36 antifungal drug resistance. To identify mechanisms underlying such instability and to uncover
37 new potential antifungal targets, we conducted an overexpression screen monitoring chromosomal
38 stability in the human fungal pathogen Candida albicans. Analysis of ~1000 genes uncovered six
39 chromosomal stability (CSA) genes, five of which are related to cell division genes in other
40 organisms. The sixth gene, CSA6, is selectively present in the CUG-Ser clade species that
41 includes C. albicans and other human fungal pathogens. The protein encoded by CSA6 localizes
42 to the spindle pole bodies, is required for exit from mitosis, and induces a checkpoint-dependent
43 metaphase arrest upon overexpression. Together, Csa6 defines an essential CUG-Ser fungal
44 clade-specific cell cycle progression factor, highlighting the existence of phylogenetically-
45 restricted cell division genes which may serve as potential unique therapeutic targets.
46

47 Teaser
48

49 Csa6 is essential for mitotic progression and mitotic exit in the human fungal pathogen Candida
50 albicans.
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67 Introduction
68

69 Cell division is a fundamental aspect of all living organisms, required to support growth,
70 reproduction and replenishment of dead or damaged cells. The primary objective of cell division
71 is to ensure genome stability by preserving and transferring the genetic material with high-fidelity
72 into progeny. Genome stability is achieved by proper execution of key cell cycle events such as
73 chromosome duplication at the S phase followed by equal segregation of the duplicated
74 chromosomes at the M phase. In addition, various cell cycle checkpoints monitor the integrity and
75 fidelity of cell cycle events in response to an error or any damage until rectified or repaired.
76 Failure of any of the error-correcting mechanisms can introduce genetic alterations, causing
77 genomic instability in progeny. Genome instability can occur as a consequence of either point
78 mutations, insertions or deletions of bases in specific genes and/or gain, loss or rearrangements of
79 chromosomes, collectively referred to as chromosome instability (CIN) (1). CIN has been
80 intimately associated with aneuploidy (2) and is one of the potential drivers of human genetic and
81 neurodegenerative disorders (3, 4), aging (5) and several cancers (6). While considered harmful
82 for a cell or an organism, CIN may also contribute to generating variations and help in driving
83 evolution, especially in unicellular primarily asexual eukaryotes (7, 8).
84

85 The current understanding of the mechanisms underlying genome stability has evolved through
86 studies in a range of biological systems from unicellular yeasts to more complex metazoa
87 including humans. These studies highlighted concerted actions of genes involved in (a) high-
88 fidelity DNA replication and DNA damage repair, (b) efficient segregation of chromosomes and
89 (c) error-correcting cellular surveillance machinery (9, 10) in maintenance and inheritance of a
90 stable genome. In recent years, large-scale screenings of loss-of-function (11), reduction-of-
91 function (12) and overexpression (13-16) mutant collections in the budding yeast Saccharomyces
92 cerevisiae have appended the list of genome stability-regulators by identifying uncharacterized
93 proteins as well as known proteins with functions in other cellular processes. However,
94 considering the vast diversity of the chromosomal segregation mechanisms in eukaryotes, it is
95 conceivable that many genes involved in genome maintenance are yet to be discovered and
96 require additional screens in a wide range of organisms for their identification. While perturbation
97 of a candidate gene’s function to decipher its role in a cellular pathway has been a classical
98 strategy in biological research, screening of strain collections aids in uncovering molecular
99 players and cellular pathways in an unbiased manner.
100

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101 The ascomycetous yeast Candida albicans is emerging as an attractive unicellular model for
102 studying eukaryotic genome biology (17). C. albicans, a commensal of both the gastrointestinal
103 and genital tracts, remains the most frequently isolated fungal species worldwide from the
104 patients diagnosed with candidemia or other nosocomial Candida infections (18, 19). The diploid
105 genome of C. albicans shows remarkable plasticity in terms of ploidy, single nucleotide
106 polymorphism (SNP), loss of heterozygosity (LOH), copy number variations, and CIN events (17,
107 20). Although LOH can be detected on all the chromosomes of C. albicans, the presence of
108 recessive lethal or deleterious alleles on some haplotypes (17), prevents one of the haplotypes or
109 even a part of it from existing in the homozygous state. In particular, this homozygous bias has
110 been observed for chromosomes R (ChR), 2 (Ch2), 4 (Ch4), 6 (Ch6) and 7 (Ch7) (21, 22). LOH
111 and aneuploidy-driven CIN has serious phenotypic consequences in C. albicans such as
112 conferring resistance to antifungals (23-28) or help C. albicans adapt to different host niches (29-
113 31). Whether genome plasticity is contributing to the success of C. albicans as a commensal
114 or/and a dreaded pathogen of humans, remains an enigma (17). Nevertheless, with increasing
115 instances of Candida infections and emerging antifungal resistance, there is an immediate need to
116 identify novel fungus-specific molecular targets that may aid the development of antifungal
117 therapies. In addition, the remarkable ability of C. albicans to tolerate CIN in the form of whole
118 chromosome loss, isochromosome formation, chromosome truncation, or mitotic crossing-over
119 (17, 20, 32) raises intriguing questions on the functioning of genome stability-regulators in this
120 fungal pathogen.
121

122 Of utmost importance to maintain genomic integrity, is the efficient and error-free segregation of
123 the replicated chromosomes. In most eukaryotes including C. albicans, the assembly of a
124 macromolecular protein complex, called the kinetochore (KT), on CENP-A (Cse4 in budding
125 yeasts) containing centromeric chromatin mediates chromosome segregation during mitosis (33-
126 35). The KT acts as a bridge between a chromosome and the connecting microtubules (MTs),
127 emanating from the spindle pole bodies (SPBs), the functional homolog of centrosomes in
128 mammals (36). The subsequent attachment of sister KTs to opposite spindle poles then promotes
129 the formation of a bipolar mitotic spindle that drives the separation of the duplicated
130 chromosomes during anaphase (37), after which cells exit mitosis and undergo cytokinesis (38-
131 40). In C. albicans, KT proteins remain clustered throughout the cell cycle and are shown to be
132 essential for viability and mitotic progression (33, 41, 42). In addition, genes involved in
133 homologous recombination, such as MRE11 and RAD50, and DNA damage checkpoint pathway,
134 including MEC1, RAD53 and DUN1, are required to prevent genome instability in C. albicans
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135 (43-45). Strikingly, aberrant expression of proteins involved in DNA damage response or cell
136 division triggers morphological transition to a unique polarized, filamentous growth in C.
137 albicans (17). A recent screen, using a collection of 124 over-expression strains, has identified
138 three additional genes, namely, CDC20, BIM1, and RAD51, with a role in genome maintenance as
139 indicated by increased LOH-driven CIN upon overexpression in C. albicans (46). Currently, only
140 a minor fraction of the C. albicans gene armamentarium has been evaluated for their roles in
141 genome stability. Systematic approaches are thus needed to exhaustively define the drivers of C.
142 albicans genome maintenance and outline species-specific processes as well as commonalities
143 with other eukaryotes.
144

145 Here, we describe a large-scale screen aimed at identifying regulators of genome stability in a
146 clinically relevant fungal model system. Our screen, involving ~20% of the C. albicans
147 ORFeome, has identified Csa6, a yet unknown player of genome stability, as a critical regulator
148 of cell cycle progression in C. albicans. Overall, this is the first-ever report of such a screen at this
149 scale in C. albicans and provides a framework for identifying regulators of eukaryotic genome
150 stability, some of which may serve as new targets for therapeutic interventions of fungal
151 infections.
152

153 Results
154

155 A reporter system for monitoring chromosome stability in C. albicans

156

157 To understand the molecular mechanisms underlying genome instability in a fungal pathogen, we
158 developed a reporter system in C. albicans in which whole chromosome loss can be distinguished
159 from other events such as break-induced replication, gene conversion, chromosome truncation or
160 mitotic crossing over (22, 46). In our prior work, a loss-of-heterozygosity (LOH) reporter strain
161 was developed for use in C. albicans (22, 46). In this strain GFP and BFP genes, linked to ARG4
162 and HIS1 auxotrophic markers, respectively, are integrated at the same intergenic locus on the left
163 arms of chromosome 4A (Ch4A) and chromosome 4B (Ch4B), respectively (Fig. 1A, S1A) (22).
164 Consequently, cells express both GFP and BFP as analyzed by flow cytometry and are
165 prototrophic for ARG4 and HIS1 genes, unless a chromosome instability (CIN) event causes loss
166 of one of the two loci (Fig. 1A, B) (22). To differentiate whole chromosome loss from other
167 events that may lead to loss of one of the two reporter loci, we modified the LOH reporter strain
168 by integrating a red fluorescent protein (RFP) reporter gene, associated with the hygromycin B
5
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169 (hyg B) resistance marker, on the right arm of Ch4B (Fig. 1A, S1A). The RFP reporter insertion is
170 sufficiently distant from the BFP locus that loss of both BFP and RFP signal (and of their linked
171 auxotrophic/resistance markers) is indicative of loss of Ch4B, rather than a localized event
172 causing loss of the BFP-HIS1 reporter insertion (Fig. 1A, S1A). Notably, while loss of Ch4A
173 cannot be tolerated due to the presence of recessive lethal alleles on Ch4B (22), loss of Ch4B
174 leads to formation of small colonies that mature into larger colonies following duplication of
175 Ch4A (46). Thus, the absence of both BFP-HIS1 and RFP-HYG B but continued presence of
176 GFP-ARG4 in the modified reporter strain, which we named as chromosome stability (CSA)
177 reporter, enables us monitor loss of Ch4B in a population. The fluorescence intensity profile of
178 GFP, BFP and RFP in the CSA reporter was validated by flow cytometry (Fig. S1B). To
179 functionally validate the CSA reporter system, we employed overexpression of CDC20, a gene
180 important for anaphase onset, activation of spindle assembly checkpoint and whose
181 overexpression is known to cause whole chromosome loss in C. albicans (46). We analyzed the
182 BFP/GFP density plots in various control strains (Fig. S1C) and monitored the loss of BFP/GFP
183 signal in cells overexpressing CDC20 (CDC20OE) by flow cytometry. As reported earlier (46), the
184 CDC20OE strain displayed a higher population of BFP +GFP- and BFP-GFP+ cells as compared to
185 the empty vector (EV) control indicating increased CIN in the CDC20OE mutant (Fig. S1D, E).
186 Next, we isolated BFP-GFP+ cells of EV and CDC20OE using flow cytometry and plated them for
187 subsequent analysis of auxotrophic/resistance markers (Fig. S1F). As noted above, upon
188 incubation of the sorted BFP-GFP+ cells, we observed the appearance of both small and large
189 colonies (Fig. S1F). Small colonies have been previously shown to be the result of loss of Chr4B
190 homolog and are predicted to be a consequence of Ch4A monosomy, eventually yielding large
191 colonies upon reduplication of Ch4A (46). We, therefore, performed the marker analysis on large
192 colonies and found that 85% of the BFP-GFP+ derived colonies of CDC20OE mutant
193 concomitantly lost both HIS1 and HYG B but retained ARG4 (Fig. S1G) suggesting the loss of
194 Ch4B homolog; flow cytometry analysis further confirmed the loss of BFP and RFP signals in
195 these colonies. The remaining 15% of colonies retained GFP-ARG4 and RFP-HYG B but not
196 BFP-HIS1 (Fig. S1G) indicating that more localized events including gene conversion, rather
197 than whole chromosome loss, were responsible for loss of the BFP signals in these cells. The
198 above data indicate that the CSA reporter system that we engineered enables precise monitoring
199 of the whole chromosome loss event in a population and enables large-scale screening of this
200 phenotype.
201

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202 Medium-throughput screening of C. albicans overexpression strains identifies regulators of

203 genome stability
204

205 Systematic gene overexpression is an attractive approach for performing large-scale functional
206 genomic analysis in C. albicans, a diploid ascomycete. Using a recently developed collection of
207 C. albicans inducible overexpression plasmids (Chauvel et al., manuscript in preparation) and the
208 CSA reporter strain described above, we generated a library of 1067 C. albicans inducible
209 overexpression strains. Each of these strains, carrying a unique ORF under control of the PTET
210 promoter, could be induced for overexpression after anhydrotetracycline (Atc) or doxycycline
211 (Dox) addition (Fig. 1C) (46, 47). To identify regulators of genome stability, we carried out a
212 primary screen with these 1067 overexpression strains by individually analyzing them for the loss
213 of BFP/GFP signals by flow cytometry (Fig. 1C, S2A, Dataset 1). Our primary screening
214 identified 23 candidate genes (out of 1067) whose overexpression resulted in ≥2-fold increase in
215 the BFP+GFP- and BFP-GFP+ population relative to the EV (Table S1, S2). Next, we carried out a
216 secondary screen with these 23 overexpression strains to revalidate the loss of BFP/GFP markers
217 by flow cytometry (Fig. 1C, S2B). As genotoxic stress is intimately linked with polarized growth
218 in C. albicans (17, 48), we microscopically examined the overexpression strains exhibiting higher
219 instability at the BFP/GFP locus during secondary screening for any morphological transition
220 (Fig. 1C, S2B). While overexpression of 17 genes (out of 23) could not reproduce the BFP/GFP
221 loss phenotype, overexpression of the six genes resulted in ≥2-fold increase in the BFP +GFP- or
222 BFP-GFP+ population as compared to the EV, with three genes (out of 6) inducing polarized
223 growth upon overexpression (Fig. S3A, B). These six genes, which we referred to as CSA genes,
224 include CSA1 (CLB4), CSA2 (ASE1), CSA3 (KIP2), CSA4 (MCM7), CSA5 (BFA1) and CSA6
225 coded by ORF19.1447 of unknown function (Fig. 1D).
226

227 Molecular mechanisms underlying CIN in CSA overexpression mutants

228

229 Out of the six CSA genes, overexpression of three genes, namely, CSA1CLB4, CSA2ASE1 and
230 CSA3KIP2 caused little or no change in the morphology of C. albicans (Fig. S3A), but triggered
231 CIN at the BFP/GFP locus, indicated by an expansion of the BFP +GFP- and BFP-GFP+ population
232 in the flow cytometry density plots (Fig. S3B, C). To further dissect the molecular mechanisms
233 leading to the loss of BFP/GFP signals in these mutants, we sorted BFP -GFP+ cells of these
234 mutants and plated them for GFP-ARG4, BFP-HIS1 and RFP-HYG B analysis, as described
235 previously for the CDC20OE mutant. We observed that a majority of the large BFP -GFP+ derived
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236 colonies of CSA1CLB4, CSA2ASE1 and CSA3KIP2 overexpression mutants lost BFP-HIS1 but retained
237 RFP-HYG B and GFP-ARG4 (Fig. S3D), suggesting that localized genome instability events,
238 rather than whole chromosome loss events, contributed to the high percentage of BFP -GFP+ cells
239 in these mutants.
240

241 Overexpression of the remaining three genes, namely CSA4MCM7, CSA5BFA1 and CSA6, drastically
242 altered the morphology of the C. albicans cells by inducing polarized/filamentous growth (Fig.
243 S3A). A connection between morphological switches and genotoxic stresses has been established
244 in the polymorphic fungus C. albicans, wherein polarized growth is triggered in response to
245 improper cell cycle regulation (41, 42, 48-50). Flow cytometric analysis of cell cycle progression
246 revealed that overexpression of CSA4MCM7, CSA5BFA1 and CSA6 shifted cells towards the 4N DNA
247 content (Fig. S3E). To further determine the cell cycle phase associated with the 4N shift, we
248 compared nuclear segregation patterns (Hoechst staining for DNA and CENP-A/Cse4 localization
249 for KT) and spindle dynamics (separation of Tub4 foci) in these overexpression mutants with
250 those of the EV control (Fig. S3F). Our results suggested the 4N shift in CSA4MCM7 and CSA6
251 overexpression mutants was a result of G2/M arrest, indicated by a high percentage of large-
252 budded cells with unsegregated DNA mass and improperly separated SPBs (Fig. S3F). In
253 contrast, the 4N shift upon CSA5BFA overexpression was a consequence of late anaphase/telophase
254 arrest, shown by an increased number of large-budded cells with segregated nuclei and SPBs (Fig.
255 S3F). Taken together, our results indicate that the polarized growth in each of CSA4MCM7,
256 CSA5BFA1 and CSA6 overexpression mutants is a probable outcome of improper cell cycle
257 progression.
258

259 Two CSA genes, namely CSA2ASE1 and CSA5BFA1, gave rise to similar overexpression phenotypes
260 in both S. cerevisiae and C. albicans (Table 1). While phenotypes related to CSA4MCM7 and CSA6
261 overexpression in S. cerevisiae or other related organisms remained unreported, the
262 overexpression phenotypes of the remaining CSA genes were along the lines of their roles in cell
263 cycle functioning, as reported in S. cerevisiae (Table 1, Fig. 1D). Altogether, our results validated
264 the role of CSA genes in regulating genome stability in C. albicans. While overexpression of
265 either CSA1CLB4, CSA2ASE1 or CSA3KIP2 induced CIN mostly through non-chromosomal loss
266 events, the effect of overexpression of either CSA4MCM7, CSA5BFA1 or CSA6 was so drastic that the
267 C. albicans mutants were arrested at different cell cycle phases with G2/M equivalent DNA
268 content (4N) and thus were unable to complete the mitotic cell cycle.
269

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270 Csa6 is an SPB-localizing protein, present across a subset of CUG-Ser clade fungal species
271

272 Among the genes identified in the screen, Csa6 was the only protein without any detectable
273 homolog in S. cerevisiae (Fig. 1D). This intrigued us to examine its presence across various other
274 fungi. Phylogenetic analysis using high confidence protein homology searches and synteny-based
275 analysis indicated that Csa6 is exclusively present in a subset of fungal species belonging to the
276 CUG-Ser clade (Fig. 2A). Strikingly, in all these species, Csa6 was predicted to have a central
277 coiled-coil domain (Fig. 2B). Epitope tagging of Csa6 with a fluorescent marker (mCherry)
278 localized it close to the KT throughout the cell cycle in C. albicans (Fig. 2C). In most unicellular
279 fungi, often found proximal to the clustered KTs, are the SPB complexes (33, 35, 51, 52).
280 Although neither the SPB structure nor its composition is well characterized in C. albicans, the
281 majority of the SPB proteins exhibit high sequence and structural conservation from yeast to
282 humans (53). Hence, we re-examined Csa6 localization with two of the evolutionarily conserved
283 SPB proteins, Tub4 and Spc110, in C. albicans (53, 54) (Fig. 2D, E). These results showed that
284 Csa6 constitutively localizes to the SPBs, close to the KTs, in cycling yeast cells of C. albicans
285 (Fig. 2D, E).
286

287 Csa6, a previously uncharacterized protein, as a key regulator of mitotic progression in C.

288 albicans
289

290 While roles of Csa6 have not been investigated before, based on our findings thus far (Fig. S3E,
291 F), we hypothesized that Csa6 plays an important function in cell cycle regulation and genome
292 stability in C. albicans. We sought to identify the molecular pathways by which Csa6 performed
293 its functions in C. albicans. We again made use of the inducible PTET promoter system to generate
294 a CSA6 OE strain (CaPJ176, PTETCSA6) in the wild-type (SN148) background of C. albicans (Fig.
295 3A). Conditional overexpression of TAP-tagged Csa6 (CaPJ181, PTETCSA6-TAP), in presence of
296 Atc, was confirmed by western blot analysis (Fig. 3B). The effect of CSA6 OE (CaPJ176,
297 PTETCSA6) on cell cycle functioning was then investigated by flow cytometric cell cycle analysis
298 (Fig. 3C) and microscopic examination of the nuclear division (Fig. 3D). As observed previously
299 (Fig. S3E, F), CSA6 OE inhibited cell cycle progression in C. albicans by arresting cells in the
300 G2/M phase, evidenced by the gradual accumulation of large-budded cells with unsegregated
301 nuclei (Fig. 3D), possessing 4N DNA content (Fig. 3C). Some of these large-budded cells also
302 underwent a morphological transition to an elongated bud or other complex multi-budded
303 phenotypes (Fig. 3D), indicating cell cycle arrest-mediated morphological switching (48) due to
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304 CSA6 OE. Strikingly, continuous upregulation of Csa6 was toxic to the cells (Fig. S4A) as nuclei
305 failed to segregate in this mutant (Fig. 3D).
306

307 Nuclear segregation during mitosis is facilitated by the formation of the mitotic spindle and its
308 dynamic interactions with chromosomes via KTs. Thus, we sought to examine both the KT
309 integrity and the mitotic spindle morphology in the CSA6 OE mutants. In C. albicans, the structural
310 stability of the KT is a determinant of CENP-A/Cse4 stability wherein depletion of any of the
311 essential KT proteins results in delocalization and degradation of the CENP-A/Cse4 by ubiquitin-
312 mediated proteolysis (50). Fluorescence microscopy and western blot analysis confirmed that
313 Cse4 was neither delocalized (Fig. S4B) nor degraded from centromeric chromatin (Fig. S4C)
314 upon CSA6 OE. Next, we analyzed the spindle integrity in CSA6 OE mutants by tagging Tub4 (SPB)
315 and Tub1 (MTs) with fluorescent proteins. Fluorescence microscopy analysis revealed that a large
316 proportion (~73%) of the large-budded cells formed an unconventional rudimentary mitotic
317 spindle structure upon CSA6 OE, wherein it had a dot-like appearance as opposed to an elongated
318 bipolar rod-like spindle structure in EV or uninduced (-Atc) strains (Fig. 3E). This suggests that
319 nuclear segregation defects in CSA6 OE mutant cells are an attribute of aberrant mitotic spindle
320 formation that might have led to the mitotic arrest.
321

322 During mitosis, surveillance mechanisms, including spindle assembly checkpoint (SAC) (55, 56)
323 and spindle positioning checkpoint (SPOC) (57, 58) operate to maintain genome stability by
324 delaying the metaphase-anaphase transition in response to improper chromosome-spindle
325 attachments and spindle misorientation, respectively. We posit that the G2/M cell cycle arrest due
326 to CSA6 OE in C. albicans could be a result of either SAC or SPOC activation. Hence, we decided
327 to inactivate SAC and SPOC, individually, in the CSA6 OE strain by deleting the key spindle
328 checkpoint genes MAD2 (41) and BUB2 (48), respectively. SAC inactivation in CSA6 OE mutant
329 cells (Fig. 4A) led to the emergence of unbudded cells with 2N DNA content (Fig. 4B, C),
330 indicating a bypass of the G2/M arrest caused by CSA6 OE. Consequently, we also observed a
331 partial rescue of the growth defect in CSA6 OE mutant cells (Fig. S5A). Next, we sought to
332 characterize the effect of SAC inactivation on the spindle integrity in CSA6 OE mutants. CSA6 OE
333 resulted in the formation of an unconventional mitotic spindle (Fig. 3E) wherein it displayed a
334 single focus of SPB (Tub4-GFP), colocalizing with a single focus of MTs (Tub1-mCherry). We
335 speculated two possibilities that may lead to the single focus of Tub4: a) a defect in the process of
336 SPB duplication or b) a delay in the separation of duplicated SPBs. Fluorescence microscopy
337 analysis revealed that SAC inactivation in CSA6 OE mutant drastically increased the percentage of
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338 large-budded cells (from ~30% to ~68%) with two separated SPB foci (Tub4-GFP) (Fig. S5B).
339 These results ruled out the possibility of an unduplicated SPB in CSA6 OE mutant cells and hinted
340 at the importance of cellular Csa6 levels for proper SPB separation and chromosome segregation
341 in C. albicans.
342

343 We next determined the effect of inactivating SPOC in the cells overexpressing Csa6. For this, we
344 generated a CSA6OE strain (CaPJ200) using the bub2 null mutant (CaPJ110) as the parent strain
345 and monitored nuclear division following Hoechst staining. Strikingly, we did not observe a
346 bypass of G2/M arrest in CSA6OE mutant upon SPOC inactivation, indicated by a persistent
347 population of large-budded cells with unsegregated nuclei (Fig. S5C). Altogether, our results
348 demonstrate that overexpression of Csa6 leads to a Mad2-mediated metaphase arrest due to a
349 malformed spindle in C. albicans.
350

351 Csa6 regulates mitotic exit network and is essential for viability in C. albicans
352

353 To further gain insights into the biological function of Csa6, we sought to generate a promoter
354 shut-down mutant of csa6 (CSA6PSD). For this, we deleted one of its alleles and placed the
355 remaining one under the control of the MET3 promoter (59) which gets repressed in presence of
356 methionine (Met/M) and cysteine (Cys/C) (Fig. 5A). Western blot analysis confirmed the
357 depletion of TAP-tagged Csa6 in CSA6PSD mutant within 6 h of growth under repressive
358 conditions (Fig. 5B). The inability of CSA6PSD mutant to grow in non-permissive conditions
359 confirmed the essentiality of Csa6 for viability in C. albicans (Fig. 5C). Subsequently, we
360 analyzed the cell cycle profile (Fig. 5D) and nuclear division dynamics (Fig. 5E) in the CSA6PSD
361 strain after a specific period of incubation in either permissive or non-permissive conditions.
362 Strikingly, Csa6 depletion, as opposed to its overexpression, resulted in cell cycle arrest at the late
363 anaphase/telophase stage, indicated by an increasing proportion of large-budded cells, possessing
364 segregated nuclei and 4N DNA content (Fig. 5D, E). Additionally, we observed cells with more
365 than two nuclei, elongated-budded cells and other complex phenotypes upon Csa6 depletion (Fig.
366 5E). While CENP-A/Cse4 remained localized to centromeres in CSA6PSD mutant as revealed by
367 the fluorescence microscopy (Fig. S6A), an increase in the cellular levels of Cse4 was observed in
368 CSA6PSD mutant by western blot analysis (Fig. S6B). The increase in Cse4 levels could be an
369 outcome of Cse4 loading at anaphase in C. albicans (60, 61). Finally, we analyzed the integrity of
370 the mitotic spindle, as mentioned previously, in CSA6PSD mutant. We noticed the mean length of
371 the anaphase mitotic spindle in Csa6-depleted cells was significantly higher (~11 µm) than that of
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372 the cells grown under permissive conditions (~6 µm), indicating a spindle disassembly defect in
373 CSA6PSD mutant (Fig. 5F).
374

375 A close link between anaphase arrest, hyper-elongated mitotic spindle and inactive mitotic exit
376 network (MEN) have been established before (40, 62, 63). Localized at the SPB, the MEN is a
377 signaling cascade in S. cerevisiae that triggers cells to come out of mitosis and proceed to
378 cytokinesis (Fig. 6A) (64). We speculated the anaphase arrest in CSA6PSD mutant could be a result
379 of an inactive MEN signaling. To determine this, we sought to bypass the anaphase arrest
380 associated with Csa6 depletion by overexpressing SOL1, the CDK inhibitor and Sic1 homolog in
381 C. albicans (65) (Fig. 6B), using the inducible PTET system mentioned previously (Fig. 6C). The
382 conditional overexpression of Protein A-tagged Sol1 upon addition of Atc was verified by
383 western blot analysis (Fig. 6D). Strikingly, SOL1OE in association with Csa6 depletion allowed
384 cells to exit mitosis but not cytokinesis, as evidenced by the formation of chains of cells with >4N
385 DNA content (Fig. 6E, F). To further examine the role of Csa6 in mitotic exit, we analyzed the
386 localization of a MEN component, Tem1, a GTPase that is known to initiate MEN signaling (39,
387 66-68). In C. albicans, Tem1 localizes to SPBs in a cell-cycle-regulated manner and is essential
388 for viability (39). Fluorescence microscopy revealed that while Tem1 is localized to both the
389 SPBs in anaphase under permissive conditions (Fig. 6G) as observed earlier (39), a high
390 percentage of Csa6-depleted cells (~78%) had Tem1 localized to only one of the two SPBs (Fig.
391 6G), suggesting an important role of Csa6 in regulating mitotic exit in C. albicans. Altogether,
392 our results demonstrate that Csa6 is required for mitotic exit and thus essential for viability in C.
393 albicans.
394

395 Csa6 of Candida dubliniensis functionally complements Csa6 of C. albicans

396

397 To further elucidate the intra-species function and localization of Csa6, we decided to ectopically
398 express Csa6 of another CUG-Ser clade species, Candida dubliniensis (CdCsa6) in C. albicans.
399 C. dubliniensis is a human pathogenic budding yeast that shares a high degree of DNA sequence
400 homology with C. albicans, and possesses unique and different centromere DNA sequences on
401 each of its eight chromosomes (69, 70). Upon protein sequence alignment, we found that CdCsa6
402 (ORF Cd36_16290) is 79% identical to Csa6 of C. albicans (CaCsa6) (Fig. 7A). The ectopic
403 expression of GFP-tagged CdCsa6 in C. albicans was carried out using the replicative plasmid
404 pCdCsa6-GFP-ARS2 (Fig. 7B), which contains the autonomously replicating sequence (ARS) of
405 C. albicans (71). Although unstable when present in an episomal form, ARS plasmids, upon
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406 spontaneous integration into the genome, can propagate stably over generations (72).
407 Fluorescence microscopy of integrated pCdCsa6-GFP-ARS2 revealed that similar to CaCsa6,
408 CdCsa6 localizes constitutively to the SPBs in C. albicans (Fig. 7C), further supporting Csa6’s
409 evolutionarily conserved role in regulating mitotic spindle and mitotic exit in C. albicans. We
410 next asked if CdCsa6 can functionally complement CaCsa6. For this, we ectopically expressed
411 CdCsa6 in CSA6PSD strain. Strikingly, the ectopic expression of CdCsa6 rescued the growth defect
412 associated with CSA6PSD mutant under non-permissive conditions, indicating CdCsa6 can
413 functionally complement CaCsa6 (Fig. 7D). This suggests functional conservation of Csa6 among
414 related Candida species belonging to the CUG-Ser clade.
415
416 Discussion
417

418 In this study, we carried out an extensive screen to identify genes that contribute to genome
419 stability in C. albicans by generating and analyzing a library of more than a thousand
420 overexpression strains. Our screen identified six regulators of chromosome stability including
421 Csa6, a protein of unknown function. Molecular dissection of Csa6 function revealed its
422 importance in cell cycle progression at least in two critical stages, metaphase-anaphase transition
423 and mitotic exit. We further demonstrated that Csa6 is constitutively localized to the SPBs,
424 essential for viability, and alterations of its cellular level leads to cell cycle arrest in C. albicans.
425 Finally, subcellular localization and complementation analysis revealed functional conservation
426 of Csa6 across the pathogenic Candida species.
427

428 The identification of two CSA genes, CSA2ASE1 and CSA5BFA1, that were earlier reported as CIN
429 genes (13, 14), further validates the power of the screening approach and the methods presented
430 in this study. The respective overexpression phenotypes of these two genes in C. albicans were
431 found to be similar to those in S. cerevisiae, suggesting that their functions might be conserved in
432 these distantly related yeast species. In S. cerevisiae, Ase1 acts as an MT-bundling protein,
433 required for spindle elongation and stabilization during anaphase (73, 74) (Fig. 8A). Hence,
434 increased CIN upon ASE1 overexpression might be an outcome of premature spindle elongation
435 and improper KT-microtubule attachments (74, 75). Bfa1, on the other hand, is a key component
436 of the Bub2-Bfa1 complex, involved in SPOC activation (57), and a negative regulator of mitotic
437 exit (76) (Fig. 8A). In S. cerevisiae, BFA1 overexpression prevents Tem1 from interacting with its
438 downstream effector protein Cdc15, thus inhibiting MEN signaling and arresting cells at the
439 anaphase (77). In our screen, a B-type mitotic cyclin Clb4 (CSA1), and a kinesin-related motor

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440 protein Kip2 (CSA3) (Fig. 8A), were found to increase CIN upon overexpression, primarily via
441 non-chromosomal loss events. C. albicans Clb4 acts as a negative regulator of polarized growth
442 (49) and is the functional homolog of S. cerevisiae Clb5 (78), required for the entry into the S-
443 phase (79). Increased CIN upon CSA1CLB4 overexpression, is thus consistent with its role in S-
444 phase initiation. The function of Kip2, however, is yet to be characterized in C. albicans. In S.
445 cerevisiae, Kip2 functions as an MT polymerase (80), with its overexpression leading to
446 hyperextended MTs and defects in SPB separation (81). The associated CIN observed upon
447 CSA3KIP2 overexpression in C. albicans is in line with its function in nuclear segregation.
448

449 Mcm7, another CSA gene (CSA4) identified in this study, is a component of the highly conserved
450 Mcm2-7 helicase complex, essential for eukaryotic DNA replication initiation and elongation (82)
451 (Fig. 8A). While Mcm7 depletion arrests cells at S phase (83), the effect of MCM7
452 overexpression on genomic integrity is comparatively less explored. Especially, several cancerous
453 cells have been shown to overexpress Mcm7 (84-86), with its elevated levels increasing the
454 chances of relapse and local invasions (84). In this study, we found that overexpression of MCM7,
455 in contrast to Mcm7 depletion, arrested cells at the G2/M stage. One possibility is increased
456 Mcm7 levels interfered with DNA replication during the S phase, resulting in DNA damage or
457 accumulation of single-stranded DNA, thus activating the RAD9-dependent cell cycle arrest at the
458 G2/M stage (87, 88). In a recent study from our laboratory, Mcm7 has been identified as a subunit
459 of the kinetochore interactome in a basidiomycete yeast Cryptococcus neoformans (89). Another
460 subunit of the Mcm2-7 complex, Mcm2, is involved in regulating the stability of centromeric
461 chromatin in C. albicans (61). Considering the growing evidence of the role of Mcm2-7 subunits
462 beyond their canonical, well-established roles in DNA replication, the serendipitous identification
463 of Mcm7 as a regulator of genome stability in our screen is striking.
464

465 We performed an in-depth analysis of Csa6, a novel regulator of cell cycle progression identified
466 from our screen (Fig. 8B, C). Our results revealed that overexpression of CSA6 leads to an
467 unconventional mitotic spindle formation and SAC-dependent G2/M cell cycle arrest (Fig. 8C) in
468 C. albicans. While mad2 deletion indicated that SPB duplication and separation of duplicated
469 SPBs is unperturbed in CSA6 overexpressing cells, what exactly triggered the activation of SAC
470 in these cells remains to be determined. Recent studies on human cell lines have shown that
471 failure in the timely separation of the centrosomes promotes defective chromosome-MT
472 attachments and may lead to chromosome lagging if left uncorrected by the cellular surveillance
473 machinery (90-92). Along the same lines, we posit that a delay in SPB separation, mediated by
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474 overexpression of Csa6, leads to increased instances of improper chromosome-MT attachments,

475 leading to SAC activation and an indefinite arrest at the metaphase stage. Future studies on the
476 SPB structure-function and composition in C. albicans should reveal how Csa6 regulates SPB
477 dynamics in this organism.
478

479 In contrast to its overexpression, Csa6 depleted cells failed to exit mitosis and remained arrested
480 at the late anaphase/telophase stage (Fig. 8C). We further linked the mitotic exit failure in Csa6
481 depleted cells with the defective localization of Tem1, an upstream MEN protein. While the
482 hierarchy of MEN components, starting from the MEN scaffold Nud1, an SPB protein, to its
483 ultimate effector Cdc14 is well established in S. cerevisiae (64), the existence of a similar
484 hierarchy in C. albicans needs to be investigated. In addition, several lines of evidence suggest
485 that MEN in C. albicans may function differently from S. cerevisiae: (a) Unlike S. cerevisiae, C.
486 albicans Cdc14 is non-essential for viability with its deletion affecting cell separation (93). (b)
487 Cdc14 is present in the nucleoplasm for the majority of the cell cycle in contrast to its nucleolar
488 localization in S. cerevisiae (93). (c) C. albicans Dbf2 is required for proper nuclear segregation,
489 actomyosin ring contraction, and cytokinesis (38). A recent study involving the identification of
490 Cdc14 interactome in C. albicans (94) found only a subset of proteins (0.2%) as physical or
491 genetic interactors in S. cerevisiae, suggesting the divergence of Cdc14 functions in C. albicans.
492 Hence, further investigations of MEN functioning in C. albicans are required to understand its
493 divergence from S. cerevisiae and the mechanism by which Csa6 regulates mitotic exit in C.
494 albicans and related species. Altogether, our results indicate that Csa6 has dual functions during
495 cell cycle progression wherein it is first required during the G2/M phase for proper assembly of
496 the mitotic spindle and then later during anaphase to exit the cells from mitosis. In addition, the
497 constitutive localization of Csa6 to the SPBs strengthens the link between SPB-related functions
498 and Csa6 in C. albicans (Fig. 8B, C).
499

500 The phylogenetic analysis of Csa6 revealed that it is only present in a group of fungal species,
501 belonging to the CUG-Ser clade. Combined with its essential cell-cycle-related functions, it is
502 intriguing to determine whether emergence of Csa6 is required to keep the pace of functional
503 divergence in the regulatory mechanisms of cell cycle progression in these Candida species.
504 While we demonstrated Csa6 of C. dubliniensis functionally complements Csa6 of C. albicans,
505 whether Csa6 of distant species can also functionally complement CaCsa6 remains to be
506 investigated. A recent study shows that around 50 essential genes, including Csa6, are only
507 present in a group of Candida species (see Dataset 5 in (95)). Identification and functional
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508 characterization of these genes in the future will aid in developing clade-specific antifungal
509 therapies (95). In this study, we have analyzed only a part of the C. albicans ORFeome for their
510 roles in genome maintenance. Further screening of the remaining overexpression ORFs will
511 provide a complete network of the molecular pathways regulating genome stability in human
512 fungal pathogens.
513

514 Materials and Methods

515

516 1. Strains, plasmids and primers. Information related to strains, plasmids and primers used in
517 this study is available in the supplementary material.
518

519 2. Media and growth conditions. C. albicans strains were routinely grown at 30°C in YPD (1%
520 yeast extract, 2% peptone, 2% dextrose) medium supplemented with uridine (0.1µg/ml) or
521 complete medium (CM, 2% dextrose, 1% yeast nitrogen base and auxotrophic supplements) with
522 or without uridine (0.1µg/ml) and amino acids such as histidine, arginine, leucine (0.1µg/ml).
523 Solid media were prepared by adding 2% agar. For the selection of transformants, nourseothricin
524 and hygromycin B (hyg B) were used at a final concentration of 100 µg/ml and 800 µg/ml,
525 respectively, in the YPDU medium.
526

527 Overexpression of genes from the tetracyline inducible promoter (P TET) was achieved by the
528 addition of anhydrotetracycline (Atc, 3 µg/ml) or doxycycline (Dox, 50 µg/ml) in YPDU medium
529 at 30°C (47) in the dark as Atc and Dox are light-sensitive. The CSA6PSD strains were grown at
530 30°C either in permissive (YPDU) or nonpermissive (YPDU + 5mM methionine (M) + 5mM
531 cysteine (C)) conditions of the MET3 promoter (59, 61). E. coli strains were cultured at 30°C or
532 37°C in Luria-Bertani (LB) medium or 2YT supplemented with ampicillin (50 µg/ml or 100
533 µg/ml), chloramphenicol (34 µg/ml), kanamycin (50 µg/ml) and tetracycline (10 µg/ml). Solid
534 media were prepared by adding 2% agar. Chemically competent E. coli cells were prepared
535 according to Chung et al (96).
536

537 3. Flow cytometry analysis. Cultures of overexpression strains following 8 h of induction in

538 YPDU+Atc and overnight recovery in the YPDU medium alone, were diluted in 1x phosphate-
539 buffered saline (PBS) and analyzed (~106 cells) for the BFP/GFP marker by flow cytometry
540 (FACSAria III, BD Biosciences) at a rate of 7000-10,000 events/s. We used 405- and 488-nm

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541 lasers to excite the BFP and GFP fluorophores and 450/40 and 530/30 filters to detect the BFP
542 and GFP emission signals, respectively.
543

544 4. Primary and secondary overexpression screening. To detect CIN at the BFP/GFP locus
545 upon PTET activation, overnight grown cultures of C. albicans overexpression strains were
546 reinoculated in CM-His-Arg to ensure all cells contained BFP-HIS1 or GFP-ARG4. To measure
547 the loss of BFP/GFP signals in 96-well plates, a CDC20OE mutant was used as a positive control.
548 The primary selection of the overexpression mutants with increased BFP +GFP- and BFP-GFP+
549 cells was done by determining the BFP/GFP loss frequency in EV. For this, we analyzed the flow
550 cytometry density plots for 22 independent cultures of EV using the FlowJo software (FlowJo X
551 10.0.7r2). We observed a similar profile for all the cultures. We then defined gates for the
552 BFP+GFP- and BFP-GFP+ fractions of cell population in one of the EV samples and applied these
553 gates to the rest of EV samples. The mean frequency of BFP +GFP- and BFP-GFP+ cells in EV was
554 calculated (Table S1). Similar gates were applied to all 1067 overexpression strains analyzed for
555 BFP/GFP markers and the frequency of BFP+GFP- and BFP-GFP+ cells for each strain was
556 determined (Dataset 1). The overexpression mutants, in which the BFP/GFP loss frequency was
557 ≥2-fold than EV, were selected for further analysis (Table S2).
558

559 For secondary screening, the overexpression plasmids present in each of the overexpression
560 strains, identified from the primary screen (23 out of 1067), were used to retransform the CSA
561 reporter strain (CEC5201). The overexpression strains (23) were analyzed by flow cytometry to
562 revalidate the loss of BFP/GFP signals. Overexpression strains displaying ≥ 2-fold higher
563 frequency of BFP+GFP- /BFP-GFP+ population than EV (6 out of 23) were monitored for any
564 morphological transition by microscopy. As filamentous morphotype could distort the BFP/GFP
565 loss analysis (46), we characterized the overexpression mutants exhibiting increased CIN at the
566 BFP/GFP locus and filamentous growth (3 out of 6) by monitoring cell cycle progression. For
567 this, we transformed the overexpression plasmids in CaPJ159 and analyzed the overexpression
568 strains (CSA4MCM7, CSA5BFA1 and CSA6) for DNA content, nuclear segregation and SPB
569 separation. The 6 genes identified from the secondary screen were verified for the correct C.
570 albicans ORF by Sanger sequencing using a common primer PJ90. During the secondary
571 screening, we also cultured overexpression mutants in YPDU without Atc and observed no
572 differences between EV and uninduced (-Atc) cultures in terms of morphology and the BFP/GFP
573 loss frequency.
574

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575 5. Cell sorting and marker analysis following a CIN event. Overnight grown cultures of EV
576 and overexpression mutants (CDC20, CSA1CLB4, CSA2ASE1 and CSA3KIP2) were reinoculated in
577 YPDU+Atc for 8 h and allowed to recover overnight in YPDU-Atc. The cultures were analyzed
578 for BFP/GFP loss by flow cytometry followed by fluorescence-activated cell sorting (FACS)
579 using a cell sorter (FACSAria III, BD Biosciences) at a rate of 10,000 events/s. Approximately
580 1500 cells from the BFP-GFP+ population were collected into 1.5-ml tubes containing 400 µl
581 YPDU and immediately plated onto YPDU agar plates. Upon incubation at 30°C for 2 days, both
582 small and large colonies appeared, as reported earlier (46). As most small colonies are expected to
583 have undergone loss of the Ch4B haplotype (46), we analyzed auxotrophic/resistance markers of
584 large colonies to characterize the molecular mechanisms underlying CIN in the overexpression
585 mutants.
586

587 For marker analysis, we replica plated the large colonies along with the appropriate control strains
588 on CM-Arg, CM-His and YPDU+hyg B (800 µg/ml) and incubated the plates at 30°C for 2 days.
589 The colonies from CM-Arg plates were then analyzed for BFP, GFP and RFP markers by flow
590 cytometry. For this, overnight grown cultures in YPDU were diluted in 1x PBS and 5000-10,000
591 cells were analyzed (FACSAria III, BD Biosciences). We used 405-, 488- and 561 nm lasers to
592 excite the BFP, GFP and RFP fluorophores and 450/40, 530/30, 582/15 filters to detect the BFP,
593 GFP and RFP emission signals, respectively.
594

595 6. Cell cycle analysis. Overnight grown cultures of C. albicans were reinoculated at an OD600 of
596 0.2 in different media (as described previously) and harvested at various time intervals post-
597 inoculation (as mentioned previously). The overnight grown culture itself was taken as a control
598 sample (0 h) for all the experiments. Harvested samples were processed for propidium iodide (PI)
599 staining as described before (33). Stained cells were diluted to the desired cell density in 1x PBS
600 and analyzed (≥30, 000 cells) by flow cytometry (FACSAria III, BD Biosciences) at a rate of
601 250-1000 events/s. The output was analyzed using the FLOWJO software. We used 561-nm laser
602 to excite PI and 610/20 filter to detect its emission signals.
603

604 7. Fluorescence microscopy. For nuclear division analysis in untagged strains, the C. albicans
605 cells were grown overnight. The next day, the cells were transferred into different media (as
606 mentioned previously) with a starting O.D.600 of 0.2, collected at various time intervals (as
607 described previously) and fixed with formaldehyde (3.7%). Cells were pelleted and washed thrice
608 with 1x PBS, and Hoechst dye (50 ng/ml) was added to the cell suspension before imaging.
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609 Nuclear division in Cse4-and Tub4-tagged strains was analyzed as described above, except the
610 cells were not fixed with formaldehyde. For Tem1 and mitotic spindle localization, overnight
611 grown cultures were transferred to different media (as mentioned previously) with a starting
612 O.D.600 of 0.2 and were grown for 6 h or 8 h. Cells were then washed, resuspended in 1x PBS and
613 imaged on a glass slide. Localization studies of each, CaCsa6, Tub4, Spc110 and CdCsa6 was
614 carried out by washing the log phase grown cultures with 1x PBS (three times) followed by image
615 acquisition.
616

617 The microscopy images were acquired using fluorescence microscope (Zeiss Axio Observer 7
618 equipped with Colibri 7 as the LED light source), 100x Plan Apochromat 1.4 NA objective, pco.
619 edge 4.2 sCMOS. We used Zen 2.3 (blue edition) for image acquisition and controlling all
620 hardware components. Filter set 92 HE with excitation 455–483 and 583–600 nm for GFP and
621 mCherry, respectively, and corresponding emission was captured at 501–547 and 617–758 nm. Z
622 sections were obtained at an interval of 300 nm. All the images were displayed after the maximum
623 intensity projection using ImageJ. Image processing was done using ImageJ. We used the cell
624 counter plugin of ImageJ to count various cell morphologies in different mutant strains. Images
625 acquired in the mCherry channel were processed using the subtract background plugin of ImageJ
626 for better visualization.
627

628 8. Protein preparation and western blotting. Approximately 3 O.D.600 equivalent cells were
629 taken, washed with water once and resuspended in 12.5% TCA (trichloroacetic acid) and
630 incubated at -20˚C overnight for precipitation. The cells were pelleted down and washed twice
631 with ice-cold 80% acetone. The pellet was then allowed to air dry and finally resuspended in lysis
632 buffer (0.1N NaOH and 1% SDS and 5xprotein loading dye). Samples were boiled at 95˚C for 5-
633 10 min and electrophoresed on a 10% SDS polyacrylamide gel. Gels were transferred to a
634 nitrocellulose membrane by semi-dry method for 30 min at 25V and blocked for an hour in 5%
635 non-fat milk in 1x PBS. Membranes were incubated with a 1:5000 dilution of rabbit anti-Protein
636 A or mouse anti-PSTAIRE in 2.5% non-fat milk in 1x PBS. Membranes were washed three times
637 in 1x PBS-Tween (0.05%) and then exposed to a 1:10,000 dilution of either anti-mouse- or anti-
638 rabbit-IgG horseradish peroxidase antibody in 2.5% non-fat milk in 1x PBS. Membranes were
639 washed three times in 1x PBS-Tween (0.05%) and developed using chemiluminescence method.
640

641 9. Statistical analysis. Statistical significance of differences was calculated as mentioned in the
642 figure legends with unpaired one-tailed t-test, paired one-tailed t-test, paired two-tailed t-test or
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643 one-way ANOVA with Bonferroni posttest. P-values ≥ 0.05 were considered as nonsignificant
644 (n.s.). P-values of the corresponding figures are mentioned, if significant. All analyses were
645 conducted using GraphPad Prism version Windows v5.00.
646

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934 Acknowledgments
935

936 We thank members of the Sanyal and d’Enfert laboratories for their valuable suggestions and
937 constructive criticism. We thank the Munro group at University of Aberdeen and Mazel group at
938 Institut Pasteur for their contribution to the establishment of overexpression plasmids that were
939 used in this study, a work that will be reported elsewhere. We thank Dr. Arshad Desai for critical
940 reading of the manuscript. We thank N. Varshney for constructing the plasmid pCse4-TAP-Leu.
941 We thank L. Sreekumar for constructing pTub4-GFP-His cassette. Special thanks to K. Guin for
28
 bioRxiv preprint doi: [Link] this version posted September 23, 2021. The copyright holder for this preprint
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available under aCC-BY-NC-ND 4.0 International license.

942 sharing the raw files to generate the phylogenetic tree. We thank V. Sood and A. Das for
943 generating the plasmid pCdCsa6-GFP-ARS2. We thank A.S. Amrutha for generating the strains
944 CaPJ300 and CaPJ301. We acknowledge N. Nala at the flow cytometry facility, JNCASR, for
945 assisting flow cytometry and cell sorting experiments. The establishment of overexpression
946 plasmids was supported by the Wellcome Trust [088858/Z/09/Z to CD]. This work was supported
947 by a grant from the Indo French Centre for the promotion of Advanced Research (CEFIPRA,
948 Project no. 5703-2). CEFIPRA also aided in the travel of PJ, KS and CD between the Sanyal and
949 d’Enfert laboratories. PJ acknowledges intramural funding from JNCASR. AD and TP were
950 supported by the CEFIPRA grant. K.S. acknowledges the financial support of JC Bose National
951 Fellowship (Science and Engineering Research Board, Govt. of India, JCB/2020/000021) and
952 intramural funding from JNCASR.
953

954 Funding
955

956 Indo French Centre for the promotion of Advanced Research (CEFIPRA, Project no. 5703-2).
957 Jawaharlal Nehru Centre for Advanced Scientific Research.
958 JC Bose National Fellowship (Science and Engineering Research Board, Govt. of India,
959 JCB/2020/000021).
960 The Wellcome Trust (088858/Z/09/Z).
961 Institut Pasteur.
962 Institut national de la recherche pour l'agriculture, l'alimentation et l'environnement (INRAE).
963

964 Author contributions:

965 Conceptualization: KS, CD, PJ, ML
966 Methodology: ML, PJ, AD, TP, MC
967 Investigation: PJ, AD, TP, ML
968 Supervision: KS, CD, ML
969 Writing—original draft: PJ, KS
970 Writing—review & editing: PJ, KS, CD, ML
971

972 Competing interests: The authors declare no competing interests.

973

974 Data and materials availability: All data are available in the main text or supplementary
975 materials.
29
 bioRxiv preprint doi: [Link] this version posted September 23, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

976 Figure 1
977

978
979

980 Fig. 1. A medium-throughput protein overexpression screen identifies a set of CSA genes in
981 C. albicans. (A) Possible outcomes of CIN at the BFP/GFP and RFP loci. 1-4, CIN at the BFP or
982 GFP locus, because of either chromosome loss (CL) or non-CL events such as break-induced
983 replication, gene conversion, chromosome truncation or mitotic crossing over, will lead to the
984 expression of either GFP or BFP expressing genes. CIN due to CL can be specifically identified
985 by the concomitant loss of BFP and RFP, as shown in 1. 5 and 6, cells undergoing non-CL events
986 at the RFP locus will continue to express BFP and GFP. (B) Flow cytometric analysis of the
987 BFP/GFP density profile of empty vector (EV) (CaPJ150) containing BFP, GFP and RFP genes.
988 Majority of the cells are positive for both BFP and GFP (BFP +GFP+). A minor fraction of the

30
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

989 population had lost either one of the markers (BFP +GFP- or BFP-GFP+) or both the markers (BFP-
990 GFP-), indicating spontaneous instability of this locus (46). Approximately 1 million events are
991 displayed. (C) Pictorial representation of the screening strategy employed for identifying CSA
992 genes in C. albicans. Briefly, a library of C. albicans overexpression strains (1067), each carrying
993 a unique ORF under the tetracycline-inducible promoter, P TET, was generated using the CSA
994 reporter (CEC5201) as the parent strain. The library was then analyzed by primary and secondary
995 screening methods to identify CSA genes. In the primary screen, CIN frequency at the BFP/GFP
996 locus in the individual 1067 overexpression strains was determined using flow cytometry.
997 Overexpression strains exhibiting increased CIN (23 out of 1067) were taken forward for
998 secondary screening. The secondary screen involved revalidation of the primary hits for increased
999 CIN at the BFP/GFP locus by flow cytometry. Strains which reproduced the increased CIN
1000 phenotype were further examined for yeast to filamentous transition by microscopy. (D) A brief
1001 overview of the CSA genes identified from the overexpression screen (6 out of 1067). Functional
1002 annotation of genes is based on the information available either in Candida Genome Database
1003 ([Link]) or in Saccharomyces Genome Database ([Link]) on
1004 August 1, 2021.
1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1023 Figure 2
1024

1025
1026

1027 Fig. 2. Csa6 has a selective existence across fungal phylogeny and is constitutively localized
1028 to the SPBs in C. albicans. (A) Phylogenetic tree showing the conservation of Csa6 across the
1029 mentioned species. The presence (filled circles) or absence (empty circles) of Csa6 in every
1030 species is marked. Each taxonomic rank is color-coded. The species mentioned under the family
1031 Debaryomycetaceae belong to the CUG-Ser clade in which the CUG codon is often translated as
1032 serine instead of leucine. The red arrow points to the CUG-Ser clade lineage that acquired Csa6.
1033 Searches for Csa6 homologs (E value ≤ 10-2) were carried out either in the Candida Genome
1034 Database ([Link]) or NCBI nonredundant protein database. (B) Schematic
1035 illustrating the protein domain architecture alignment of Csa6 in the indicated fungal species.

32
 bioRxiv preprint doi: [Link] this version posted September 23, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1036 Length of the protein is mentioned as amino acids (aa). Approximate positions of the predicted
1037 coiled-coil domain, identified using HMMER (97) phmmer searches, is shown. (C-E) Left,
1038 micrographs comparing the sub-cellular localization of Csa6 with KT (Cse4) and SPB (Tub4 and
1039 Spc110) at various cell cycle stages. Top, Csa6-mCherry and Cse4-GFP (CaPJ119); middle, Csa6-
1040 mCherry and Tub4-GFP (CaPJ120), and bottom, Csa6m-Cherry and Spc110-GFP (CaPJ121).
1041 Scale bar, 1 µm. Right, histogram plots showing the fluorescence intensity profile of Csa6-
1042 mCherry with Cse4-GFP (top), Tub4-GFP (middle) and Spc110-GFP (bottom) across the
1043 indicated lines.
1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1066 Figure 3

1067
1068

34
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1069 Fig. 3. Overexpression of Csa6 alters the morphology of the mitotic spindle and leads to
1070 G2/M arrest in C. albicans. (A) Atc/Dox-dependent functioning of the PTET promoter system for
1071 conditional overexpression of CSA6. (B) Western blot analysis using anti-Protein A antibodies
1072 confirmed overexpression of CSA6-TAP from the PTET promoter (CaPJ181), after 8 h induction in
1073 presence of Atc (3 µg/ml), in comparison to the uninduced culture (-Atc) or CSA6-TAP
1074 expression from its native promoter (CaPJ180); N=2. PSTAIRE was used as a loading control.
1075 UT, untagged control (SN148). (C) Flow cytometric analysis of cell cycle displaying the cellular
1076 DNA content of CSA6 OE strain (CaPJ176) in presence or absence of Atc (3 µg/ml) at the
1077 indicated time intervals; N=3. (D) Left, microscopic images of Hoechst-stained EV (CaPJ170)
1078 and CSA6OE strain (CaPJ176) after 8 h of growth under indicated conditions of Dox (50 µg/ml).
1079 BF, bright-field. Scale bar, 10 µm. Right, quantitation of different cell types at the indicated time-
1080 points; n ≥100 cells. (E) Top, representative micrographs of spindle morphology in the large-
1081 budded cells of EV (CaPJ172) and CSA6 OE strain (CaPJ178) after 8 h of growth under indicated
1082 conditions of Dox (50 µg/ml). SPBs and MTs are marked by Tub4-GFP and Tub1-mCherry,
1083 respectively. Scale bar, 1 µm. Bottom, the proportion of the large-budded cells with indicated SPB
1084 phenotypes; n ≥100 cells.
1085

1086

1087

1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

1101

1102

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1103 Figure 4
1104

1105
1106

1107 Fig. 4. The G2/M cell cycle arrest in the CSA6 OE mutant is mediated by Mad2. (A) The
1108 G2/M arrest posed by SAC in response to an improper chromosome-spindle attachment is
1109 relieved in the absence of Mad2, allowing cells to transit from metaphase to anaphase. (B) Flow
1110 cytometric DNA content analysis in CaPJ176 (MAD2CSA6 OE) and CaPJ197 (mad2CSA6 OE) at
1111 the indicated times, in presence or absence of Atc (3 µg/ml); N=3. (C) Left, microscopic images
1112 of CaPJ170 (EV), CaPJ176 (MAD2CSA6OE) and CaPJ197 (mad2CSA6 OE) following Hoechst
1113 staining, after 8 h of growth under indicated conditions of Dox (50 µg/ml). Scale bar, 10 µm.
1114 Right, quantitation of the indicated cell types; n ≥100 cells.

36
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1115 Figure 5
1116

1117
1118

1119 Fig. 5. Csa6 depletion causes late anaphase/telophase arrest with a hyper-extended mitotic
1120 spindle in C. albicans. (A) The MET3 promoter system for depleting cellular levels of Csa6. The
1121 MET3 promoter can be conditionally repressed in presence of methionine (Met/M) and cysteine
1122 (Cys/C). (B) Western blot analysis using anti-Protein A antibodies revealed time dependent
1123 depletion of Csa6-TAP in CSA6PSD strain (CaPJ212), grown under repressive conditions (YPDU
1124 + 5 mM Met and 5 mM Cys) for indicated time interval; N=2. (C) Csa6 is essential for viability in
1125 C. albicans. Strains with indicated genotypes, (1) SN148, (2) CaPJ209, (3 and 4) CaPJ210 (two

37
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1126 transformants) were streaked on agar plates with permissive (YPDU-Met-Cys) or repressive
1127 (YPDU + 5 mM Met and 5 mM Cys) media and incubated at 30°C for two days. (D) Cell cycle
1128 analysis of CaPJ210 (CSA6PSD) by flow cytometry under permissive (YPDU-Met-Cys) and
1129 repressive conditions (YPDU + 5 mM Met and 5 mM Cys) at the indicated time intervals; N=3.
1130 (E) Left, microscopic images of Hoechst stained CaPJ210 (CSA6PSD) cells grown under
1131 permissive (YPDU-Met-Cys) or repressive (YPDU + 5 mM Met and 5 mM Cys) conditions for 6
1132 h. BF bright-field. Scale bar, 5 µm. Right, quantitation of different cell types at the indicated time-
1133 points; n ≥100 cells. (F) Left, micrograph showing Tub4-GFP and Tub1-mCherry (representing
1134 mitotic spindle) in the large-budded cells of CaPJ211 (CSA6PSD) after 6 h of growth under
1135 permissive (YPDU-Met-Cys) or repressive (YPDU + 5 mM Met and 5 mM Cys) conditions.
1136 Scale bar, 3 µm. Right, quantitation of the distance between the two SPBs, along the length of the
1137 MT (representing spindle length), in large-budded cells of CaPJ211 (CSA6PSD) under permissive
1138 (n=32) or repressive (n=52) conditions. Paired t-test, one-tailed, P-value shows a significant
1139 difference.
1140

1141

1142

1143

1144

1145

1146

1147

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

38
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1160 Figure 6

1161

39
 bioRxiv preprint doi: [Link] this version posted September 23, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1162 Fig. 6. Csa6 is required for mitotic exit in C. albicans. (A) The MEN components in S.
1163 cerevisiae. At SPB, Nud1 acts as a scaffold. The ultimate target of the MEN is to activate Cdc14
1164 phosphatase, which remains entrapped in the nucleolus in an inactive state until anaphase. Cdc14
1165 release brings about mitotic exit and cytokinesis by promoting degradation of mitotic cyclins,
1166 inactivation of mitotic CDKs through Sic1 accumulation and dephosphorylation of the CDK
1167 substrates (64). (B) Inhibition of the MEN signaling prevents cells from exiting mitosis and
1168 arrests them at late anaphase/telophase. Bypass of cell cycle arrest due to the inactive MEN, viz.
1169 by overexpression of Sic1-a CDK inhibitor, results in the chain of cells with multiple nuclei (98,
1170 99). (C) A combination of two regulatable promoters, P TET and PMET3, was used to overexpress C.
1171 albicans homolog of Sic1, called SOL1 (Sic one-like), in Csa6-depleted cells. The resulting strain,
1172 CaPJ215, can be conditionally induced for both SOL1 overexpression upon Atc/Dox addition and
1173 Csa6 depletion upon Met (M)/Cys (C) addition. (D) Protein A western blot analysis showed
1174 increased levels of Sol1 (TAP-tagged) in the SOL1OE mutant (CaP217, PTETSOL1-TAP) after 6 h
1175 induction in presence of Atc (3 µg/ml) in comparison to the uninduced culture (-Atc) or SOL1
1176 expression from its native promoter (CaPJ216, SOL1-TAP); N=2. PSTAIRE was used as a
1177 loading control. UT, untagged control (SN148). (E) Flow cytometric analysis of cell cycle
1178 progression in CaPJ215 at indicated time intervals under various growth conditions, as indicated;
1179 N=3. Dox: 50 µg/ml, Met: 5 mM, Cys: 5 mM. (F) Left, Hoechst staining of CaPJ215 after 6 h of
1180 growth under indicated conditions of Dox (50 µg/ml), Met (5 mM) and Cys (5 mM); n ≥100 cells.
1181 BF bright-field. Scale bar, 5 µm. Right, percent distribution of the indicated cell phenotypes; n
1182 ≥100 cells. (G) Left, co-localization analysis of Tem1-GFP and Tub4-mCherry in large-budded
1183 cells of CaPJ218 (CSA6PSD) under permissive (YPDU-Met-Cys) or repressive conditions (YPDU
1184 + 5 mM Met and 5 mM Cys). Scale bar, 3 µm. Right, the proportion of the large-budded cells
1185 with indicated Tem1 phenotypes; n ≥100 cells.
1186

1187

1188

1189

1190

1191

1192

1193

1194

1195

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1196 Figure 7
1197

1198
1199

1200 Fig. 7. Ectopic expression and functional conservation of CdCsa6 in C. albicans. (A) Pair-
1201 wise alignment of amino acid sequences of Csa6 proteins in C. albicans (CaCsa6) and C.
1202 dubliniensis (CdCsa6) by Clustal Omega, visualized using Jalview. (B) Ectopic expression of
1203 CdCsa6 in C. albicans by random genomic integration of the ARS-containing plasmid. Vector
1204 map of pCdCsa6-GFP-ARS2 depicts the cloned sites of CaURA3, CaARS2 and CdCSA6-GFP.
1205 The CdCSA6-GFP fragment contains the GFP tag, CdCSA6 (ORF Cd36_16290) without the stop
1206 codon and the promoter region of CdCSA6. (C) CdCsa6 localizes to the SPB. Representative
1207 micrographs showing CdCsa6GFP localization at different cell cycle stages in CaPJ300.
1208 Tub4mCherry was used as an SPB marker. Scale bar, 3 µm. (D) CdCsa6 functionally
1209 complements CaCsa6. Strains with indicated genotypes, (1) SN148, (2) CaPJ300, (3) CaPJ301
1210 and (4) CaPJ302, were streaked on agar plates with permissive (YPDU-Met-Cys) or repressive
1211 (YPDU + 5 mM Met and 5 mM Cys) media and incubated at 30°C for two days.
1212

1213

1214

1215

41
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1216 Figure 8
1217

1218
1219

1220 Fig. 8. Csa6 levels are fine-tuned at various stages of the cell cycle to ensure both mitotic
1221 progression and mitotic exit in C. albicans. (A) A diagram illustrating the functions of the
1222 identified CSA genes except CSA6 in various phases and phase transitions of the cell cycle. (B)
1223 Schematic depicting the approximate position of Csa6 with respect to SPB and KT. In C.
1224 albicans, SPBs and clustered KTs remain in close proximity throughout the cell cycle, while Csa6
1225 remains constitutively localized to the SPBs. (C) A model summarizing the effects of
1226 overexpression or depletion of Csa6 in C. albicans. A wild-type cell with unperturbed Csa6 levels

42
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available under aCC-BY-NC-ND 4.0 International license.

1227 progresses through the mitotic cell cycle. Overexpression of CSA6 alters the mitotic spindle
1228 dynamics which might lead to improper KT-MT attachments, prompting SAC activation and
1229 G2/M arrest. In contrast, decreased levels of Csa6 inhibit the MEN signaling pathway, probably
1230 by affecting Tem1 recruitment to the SPBs, resulting in cell cycle arrest at the anaphase stage.
1231

1232

1233

1234

1235

1236

1237

1238
1239
1240

1241

1242

1243

1244

1245

1246

1247

1248

1249

1250

1251

1252

1253

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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

1254 Table 1. Overexpression phenotypes of CSA genes in C. albicans and S. cerevisiae

1255

S. Overexpression Overexpression
CSA C. albicans
cerevisiae phenotype (C. phenotype (S. Reference
gene ORF no.
homolog albicans) cerevisiae)
Increased CIN Shift towards 2N
CSA1 19.7186 CLB4 involving non-CL (diploid) DNA content (100)
events
i) CIN involving loss
of an artificial
chromosome fragment
or rearrangements/
gene conversion
Increased CIN
events.
CSA2 19.7377 ASE1 involving non-CL (14, 75)
events
ii) Spindle checkpoint
dependent delay in
entering anaphase
upon HU treatment

Increased CIN
Shift towards 2N
CSA3 19.1747 KIP2 involving non-CL (81, 100)
(diploid) DNA content
events
Shift towards 4N
(diploid) DNA
CSA4 19.202 MCM7 NA NA
content, G2/M
arrest
Shift towards 4N
Shift towards 2N
(diploid) DNA
CSA5 19.608 BFA1 (diploid) DNA content, (101)
content, anaphase
Anaphase arrest
arrest
Shift towards 4N
(diploid) DNA
CSA6 19.1447 NA NA NA
content, G2/M
arrest
1256 NA, not available
1257

1258

1259

1260

1261

1262

1263

1264

1265
44

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