Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.


Introduction
Most cells under unaltered conditions of growth are able to maintain their size within a strict range, and a current view sustains that the cell cycle and cell growth machineries should be interconnected by specific molecular mechanisms ensuring cell size homeostasis [1][2][3][4][5]. Budding yeast cells control their size mainly at Start [6,7], when a G1 cyclin, Cln3, acts as the most upstream activator [8]. Cyclin Cln3 forms a complex with Cdc28, the cell cycle Cdk in budding yeast, which phosphorylates the transcriptional inhibitor Whi5 and induces a transcriptional wave in circa 200 genes to trigger cell cycle entry [9]. Cln3 modulates cell volume at Start in a precise, dose-dependent manner [10][11][12], which suggests that mechanisms regulating its levels or activity likely play important roles in cell size determination. In this regard, Cln3 is present at low and nearly constant amounts throughout G1 [8, but see 13,14], and its nuclear levels are restrained by retention at the ER [15,16] and ubiquitin-mediated degradation by the proteasome [17,18].
It has long been known that cell size increases linearly with ploidy in fungi [19][20][21], plants [22], and animals [23,24], a function that is maintained across the enormous DNA content variation among eukaryotes [25] and has been used to infer ploidy in the fossil record [26]. Although ploidy has direct implications in cell growth and development, the underlying mechanisms that set cell size as a function of ploidy remain elusive [27]. Here, we describe a pathway linking the centromere (CEN) to the Start network in budding yeast. Briefly, we have found that an excess number of CENs increases degradation of Cln3 in the nucleus by a mechanism that involves physical and functional interactions between Cdc4, the specific F-box protein that targets Cln3 to SCF for ubiquitination, and Mad3, a centromeric signaling protein.

Results and discussion
In control experiments in which the size of yeast cells was carefully measured, we had previously observed that the presence of an empty yeast centromeric plasmid (YCp) produced a slightly larger volume at budding. Interestingly, this effect was exacerbated by increasing the number of empty centromeric vectors with different auxotrophic markers, suggesting that G1 length could be modulated by a genetic determinant present in these extrachromosomal DNA molecules. After ruling out possible effects due to plasmid-borne auxotrophic markers (S1A Fig), we analyzed newborn daughter cells during cell cycle entry in time-lapse experiments and found that, while initial volume was very similar, YCp caused a strong delay in G1 and a larger cell size at budding (S1B and S1C Fig). To assess the effects of YCp copy number at the single-cell level, we inserted a TEF1p-driven transcription unit expressing green fluorescent protein (GFP) in YCp vectors and mCherry in chromosome 5 and used different approaches to increase the number of centromeric sequences in the cell, some of them in a conditional manner (Fig 1A). We first analyzed cells in the simplest scenario, i.e., containing three GFPexpressing YCp vectors. Budding volume of control cells displayed a large variability [21,28] but steadily increased with the GFP/mCherry ratios (Fig 1B, see S1 Data for a detailed statistical analysis). Intriguingly, the observed trend was compatible with the doubling in budding volume displayed by diploid cells. A yeast episomal plasmid (YEp), which is present at much higher copy numbers, did not significantly alter budding volume (Fig 1C), thus pointing to the autonomous-replicating sequence (ARS) or the CEN as the YCp-specific genetic determinants modulating cell size at budding. To discern between these possibilities, we used a yeast CEN placed immediately downstream from the inducible GAL1 promoter as a conditional CEN that, by growing cells under conditions that activate (galactose) or repress (glucose) transcription from the GAL1 promoter, can be switched off or on, respectively [29]. We introduced this conditional CEN (Fig 1A) into three different YCp vectors and observed that, under permissive conditions, cell volume at budding increased with a much steeper slope compared to unmodified YCp ( Fig 1D). To rule out possible topological effects due to the circular conformation of YCp vectors, we used a linear yeast artificial chromosome (YAC) containing a conditional CEN to obtain a wide range of copy numbers per cell. As shown in Fig 1E, budding volume correlated with YAC copy number in a similar manner to that obtained with YCp vectors. Moreover, as this effect was also observed with a circular YAC derivative (S2 Fig), we were able to rule out possible additional effects of telomeric sequences. Finally, introducing conditional CENs into chromosomes 4 and 7 caused a significant increase in the budding volume of newborn daughter cells obtained by differential gradient centrifugation when allowed to enter the cell cycle under permissive conditions ( Fig 1F). As previously described [30,31], high copies of centromeric vectors caused a short mitotic delay (S3A Fig) that depended on the spindle-assembly checkpoint (SAC) [32,33]. However, this delay was much shorter than that observed during cell-cycle entry (S1C Fig), suggesting that elevated CEN copies have a greater impact in G1. Accordingly, additional CEN sequences caused a small but significant increase in the proportion of cells in G1 phase in asynchronous cultures (S3B Fig). Overall, these data indicate that CEN number modulates G1 length in daughter cells and regulates their size at budding.
Budding yeast cells mainly determine their size at Start [4]. Thus, we reasoned that signals originating from the CEN could target specific components of the Start network. YCp vectors clearly increased budding volume in cells deficient in Whi5 (Fig 2A and 2B), thus ruling out this transcriptional repressor of the G1/S regulon [34,35]. By contrast, cells lacking Cln3, the most upstream G1 cyclin [8,10,36] acting at Start, did not increase their size further, indicating that Cln3 is essential in the mechanisms that allow centromeric signals to modulate cell size. Overexpression of wild-type Cln3, which causes a strong nuclear accumulation of this G1 cyclin [15,37], also suppressed the YCp-mediated effects on budding size. However, a Cln3-1 hyperstable mutant that also reaches high levels but lacks the C-terminal nuclear-localization signal (NLS) that is essential for nuclear import of Cln3 [38] was as sensitive as wild type to the presence of YCp (Fig 2A and 2B). Supporting the notion that centromeric-dependent effects take place in the nucleus, a different hyperstable Cln3 ΔPEST mutant protein that retains the Cterminal NLS and strongly accumulates in the nucleus [15,37] fully suppressed YCp-mediated effects in cell size. Together, our data point to the idea that centromeric-dependent signals target, directly or indirectly, the yeast G1 cyclin in the nucleus.
A high-throughput two-hybrid analysis in budding yeast [39] had revealed an interaction between the Cln3 cyclin and Mad3, a component of the kinetochore-signaling network involved in the SAC [40,41]. Thus, we tested whether centromeric signaling proteins could have a role in modulating budding size as a function of YCp copy number (Fig 3A). The budding size of cells lacking either Mad3 or Bub3 was absolutely refractory to increasing copies of YCp while, contrarily, kinase Bub1 did not have any effect. These results suggest that Mad3/ Bub3 inhibit Cln3 function in a Bub1-independent manner, thus defining a mechanism different to that executing the SAC.
Newborn daughter haploid and diploid cells lacking Mad3 displayed a smaller volume at budding compared to wild type ( Fig 3B). However, size reduction was only moderate compared to the difference between haploid and diploid wild type, and mad3 cells reduced their size normally from diploid to haploid status. These data suggest that either Mad3 is not required per se in the sensing mechanism or cells must have additional or backup mechanisms to adjust cell size to ploidy (see below). Although Mad3 could not be overexpressed to much higher levels compared to the endogenous copy (S4A and S4B Fig), budding size displayed a clear increase under these mild overexpression conditions (Fig 3B). Considered together, these data reinforce the notion of an inhibitory role for Mad3 in cell cycle entry and cell size determination at budding.
Mad proteins use different but complementary mechanisms to modulate degradation of Cdc20 targets by the anaphase-promoting complex (APC/Cdc20), including mitotic cyclins [32,33], which suggests that the Mad3-dependent effects of YCp vectors on budding volume could be mediated by degradation of Cln3. Supporting this idea, Skp1 is a highly expressed centromeric protein that is also present in SCF, the E3 ubiquitin ligase required to degrade Cln3 [18]. We found that the presence of YCp vectors strongly increased the degradation rate of Cln3 in promoter shut-off experiments, and more importantly, this effect required Mad3 To support these findings further, we used a partially hyperstable and hypoactive mutant (Cln3-11A) fused to mCitrine that has no gross effects on cell cycle progression [42] but allows detection of this cyclin in G1 cells by fluorescence microscopy to monitor Cln3 degradation specifically in the nucleus. Although cells expressing mCitrine-Cln3-11A displayed an increased volume at budding when compared to wild-type cells, the presence of YCp vectors caused a similar relative increment in their budding size (S6A Fig), which validated its use. Notably, by measuring mCitrine-Cln3-11A levels in G1 cells after cycloheximide addition, we found that the presence of YCp vectors also increased the degradation rate of this G1 cyclin in the nucleus in a Mad3-dependent manner (Fig 4C and  S6B Fig). Accordingly, mCitrine-Cln3-11A steady-state levels were strongly decreased by YCp in the nucleus of G1 cells within the same volume range (Fig 4D). Since Cln3 is rate-limiting for triggering Start and setting the critical size at budding, these results would explain why the presence of YCp vectors causes a larger cell size. Next we analyzed the interaction between Mad3 and Cln3 by affinity purification and found that they yielded relative coprecipitation efficiencies similar to Cln3 and Cdc4ΔFbox (Fig 4E), the adaptor protein that recruits Cln3 to SCF in the nucleus [18]. Interestingly, we were able to detect an interaction between Cdc4 and Mad3 (Fig 4F), which suggests that Mad3 is present with Cdc4 in SCF complexes. Mad3 contains a GLEBS domain that is known to interact with Bub3 and, as a likely consequence, with Skp1 [43], and we found that Mad3 lacking the GLEBS domain does not efficiently interact with either Cdc4 or Cln3 (Fig 4E and 4F). Finally, modulation of budding size as a function of YCp copy number was strongly dampened by deleterious SCF mutations or deletion of the Mad3 GLEBS domain (Fig 4G and S7 Fig), supporting the essential role of a SCF-Cdc4/Mad3 complex in boosting Cln3 degradation to modulate cell size at budding as a function of CEN copy number.
In summary, we have uncovered a pathway that links centromeric signaling proteins to G1 cyclin stability and, hence, cell size determination in budding yeast (Fig 5). SCF-Cdc4 is estimated to be at low levels in the nucleus of yeast cells, and we envisage that Mad3, which is present at much higher levels, could act as a co-adaptor to increase the affinity of Cdc4 for Cln3. Strikingly, Cdc4 and Cdc20 display a high degree of similarity (34.2%) and contain WD40 segments that are used to interact with client proteins. However, the interaction of Mad3 would have different outcomes: (1) prevent Cdc20 from binding its targets in metaphase and (2) acting as an adaptor bridging Cln3 to Cdc4 in G1.
Mad3 is present at rather constant levels throughout the cell cycle [41], and Spc105, a scaffold protein involved in Mad3 activation at kinetochores by SAC [32], is already present in CENs in G1 [44]. Thus, by mechanisms different from those operating the SAC, Mad3 could be specifically activated in G1 at the kinetochore and sustain degradation of Cln3 at levels proportional to the number of CENs during G1 progression. Alternatively, we would like to speculate that the pathway uncovered here could belong to a Mad3-dependent checkpoint triggered by the excess of a kinetochore component that, being synthesized as a function of cell mass, would act as ploidy-mass reporter. Since Mad3-deficient or overexpressing cells do not display strong alterations in cell size, Mad3 would have a role as an effector of the checkpoint, not as sensor. Furthermore, the uncovered mechanism could be used to ensure that CENs congregate at the spindle-pole body (SPB) [45] before cell-cycle entry in budding yeast. While structural determinants of centromeric DNA are strikingly different in yeast and mammalian cells, kinetochore structural and signaling proteins are very well conserved. For this reason, we envisage that the mechanism operating in budding yeast could also exist across the evolutionary scale.
Previously proposed mechanisms to adjust cell size to ploidy [3] have not received sufficient experimental support. Although Whi5 is expressed at levels that depend on ploidy [42], diploid cells lacking one WHI5 copy are larger than haploid wild-type cells. On the other hand, introduction of additional Cln3-targeted promoters delays cell-cycle entry and increases cell size at budding [46]. However, it remains unclear whether titration of Cln3 by genome duplication is sufficient to produce a diploid cell size. We propose that, most likely with the contribution of these mechanisms, CEN-dependent degradation of Cln3 may play a pivotal role in scaling size with ploidy, a universal property of cells.

Time-lapse microscopy
Yeast cells were analyzed by time-lapse microscopy in 35-mm glass-bottom culture dishes (GWST-3522, WillCo) essentially as described [28] using a fully-motorized Leica AF7000 microscope. Time-lapse images were analyzed with the aid of BudJ, an ImageJ (Wayne Rasband, NIH) plugin that can be obtained from www.ibmb.csic.es\home\maldea to obtain cell dimensions and fluorescence levels in cellular and nuclear compartments [28]. Briefly, cell boundaries are detected as pixels markedly darker compared to both the surrounding background and the cell interior. Once outliers have been removed, an ellipse is fitted to the obtained boundary pixel array, and major and minor axes are used to calculate the cell volume assuming a prolate as shape. The same cell is followed through consecutive time-lapse images by using the center of the ellipse as seed point to obtain radial profiles in the following image.

Miscellaneous
Small daughter cells were isolated from Ficoll gradients as described [52]. DNA content distributions were obtained by Fluorescence Activated Cell Sorting [51].

Statistical tests
Pairwise comparisons were performed with non-parametric tests. Specifically, median cell volumes at budding were compared with a Mann-Whitney U test. On the other hand, correlation of cell volume at budding with GFP/mCherry ratios was analyzed with a Spearman rank test. For pairwise analysis, data were subject to bootstrap resampling (N = 100), and the resulting median slopes were compared by a Mann-Whitney U test. For both median and regression analysis, the resulting p values are shown in the corresponding figure panels. Cells carrying a YAC-CEN GALp artificial circular chromosome with no telomeric sequences were grown at restrictive conditions for the conditional CEN GALp CEN to obtain a wide range of copies per cell, returned to permissive conditions and analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small gray dots) were binned, and mean values (large orange circles, N = 50) and a regression line are plotted. The mean budding size for wild-type diploid cells is also plotted (black diamond). Nonparametric correlation analysis was performed as described in Materials and methods. Underlying data can be found in S1 Data. CEN, centromere.  Fig 1B at the restrictive temperature for cdc53ts and cdc34ts alleles to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. Correlation pairwise comparisons were performed with a nonparametric test as described in Materials and methods. Underlying data can be found in S1 Data. CEN, centromere; YCp, yeast centromeric plasmid. (TIF) S1 Data. Source data for all plots in manuscript. (XLSX)