Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 ± 4% of the GABAergic synapses and 25 ± 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.We have recently developed a method for the preparation of a brain fraction enriched in GABAergic postsynaptic complex (1). This fraction, insoluble in Triton X-100, was enriched in Gray''s type-II postsynaptic densities (type-II PSDs)² and in the postsynaptic GABAergic markers GABA_A receptors (GABA_ARs) and gephyrin. Here we report that septin 11 is a major component of the type-II PSD fraction.Septins are a family of proteins with GTPase activity that form heterooligomeric filaments and ringlike structures that act as diffusion barriers and scaffolds. Septins are involved in cytokinesis, positioning of the mitotic spindle, cellular morphology, vesicle trafficking, apoptosis, neurodegeneration, and neoplasia (2–5). In mammals, 14 septin genes have been identified. Each septin gene is expressed in several spliced forms. Although most septins are highly expressed in the brain (6), only recently is their role in neuronal function (7–9) and in neuropathology (10–14) is beginning to be addressed for some septins.Septin 11 is expressed in various tissues, including the brain (15), but little is known about the role of septin 11 in the brain. Septins 3, 5, 6, and 7 are localized in the presynaptic terminals, frequently associated with synaptic vesicles (6, 16, 17). In neurons, septin 11 forms heterooligomeric complexes with septin 7 and septin 5 (9, 18). Nevertheless, the regional and developmental distribution of septin 11 in the brain and in hippocampal cultures is not identical to that of septin 7 or septin 5 (8). These results and other heterooligomerization studies show that septin 11 is not always associated with septin 7 and septin 5 (7, 15, 19). Thus, septin 11 is expected to have functional properties both similar to and different from those of septin 7 and other septins that heterooligomerize with septin 11. In the present paper, we show that septin 11 is associated with the GABAergic synapses, particularly with the postsynapse, and concentrates at the neck of dendritic spines in the intact brain. Others have recently shown that another septin (septin 7) accumulates at the base of dendritic protrusions of cultured neurons (8, 9). However, it is not known whether septins also accumulate at the base of the dendritic spines in the brain. To the best of our knowledge, this is the first time that (i) a septin has been shown to be associated with GABAergic synapses and (ii) a septin has been shown to concentrate at the neck of dendritic spines and dendritic branching points in the intact brain.     

Septins recognize micron-scale membrane curvature

How cells recognize membrane curvature is not fully understood. In this issue, Bridges et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512029) discover that septins, a component of the cytoskeleton, recognize membrane curvature at the micron scale, a common morphological hallmark of eukaryotic cellular processes.Eukaryotic cells have dedicated proteins that sense membranes, depending on their curvature (Antonny, 2011). Sensors of membrane curvature are important because they organize a wide variety of cellular functions, including vesicle trafficking and organelle shaping (McMahon and Gallop, 2005). Curvature-sensing proteins, for example, the Bin-Amphiphysin-Rvs (BAR) domain–containing proteins, have been mostly described to work at the nanometer scale (Zimmerberg and Kozlov, 2006). In contrast, a clear mechanism of sensing membrane curvature at the micron scale in eukaryotic cells has not been described. In this issue, Bridges et al. discover that septins, a poorly understood component of the cytoskeleton, recognize plasma membrane curvature at the micron scale and serve as landmarks for eukaryotic cells to know their local shape.Septins are an evolutionarily conserved family of GTP-binding proteins that assemble into nonpolar filaments and rings (John et al., 2007; Sirajuddin et al., 2007; Bertin et al., 2008). Septins have been implicated in diverse membrane organization events where micron-scale curvature takes place (Saarikangas and Barral, 2011; Mostowy and Cossart, 2012), including the cytokinetic furrow, the annulus of spermatozoa, the base of cellular protrusions (e.g., cilium and dendritic spines), and the phagocytic cup surrounding invasive bacterial pathogens (Fig. 1). However, the precise role of septin–membrane interactions remains elusive. It was first suggested in 1999 that the interaction of human septins with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is important for septin localization (Zhang et al., 1999). More recently, work using recombinant septins from budding yeast Saccharomyces cerevisiae assembled on PI(4,5)P2 lipid monolayers showed that septins interact with membrane to facilitate filament assembly (Bridges et al., 2014). Membrane-facilitated septin assembly has also been observed using phospholipid liposomes, and in this case septins were also shown to induce membrane tubulation (Tanaka-Takiguchi et al., 2009). Given that (a) septins can interact with membrane, (b) septin assembly is membrane facilitated, and (c) septin assemblies are associated with a variety of membrane organization events from yeast to mammals, Bridges et al. (2016) hypothesized that septins serve as a mechanism to recognize membrane curvature.Open in a separate windowFigure 1.Morphological hallmarks of eukaryotic cells characterized by micron-scale membrane curvature and septin assembly. Septins have been implicated in membrane organization events where micron-scale curvature takes place. (A) A septin ring acts as a scaffold for cytokinesis proteins and forms a diffusion barrier at the cytokinetic furrow of a dividing cell. (B) A septin ring forms a diffusion barrier at the annulus of a mammalian spermatozoon, which separates the anterior and posterior tail. (C) A septin ring forms a diffusion barrier at the base of a cilium to separate the ciliary membrane from the plasma membrane. (D) In neurons, a septin-dependent diffusion barrier can localize at the base of dendritic spine necks. (E) During phagocytosis, a cup is formed at the plama membrane; septin rings assemble at the base of the phagocytic cup to regulate entry.In their new work, Bridges et al. (2016) provide several lines of evidence to support the hypothesis that septins recognize micron-scale curvature. First, using the filamentous fungus Ashbya gossypii, they performed in vivo localization studies and showed that the fungal septin Cdc11a concentrates in regions of positive micron-scale curvature and that the degree of concentration is proportional to the degree of curvature. Moreover, septins localize to curved membranes that also recruit septin-interacting proteins (e.g., the signaling protein Hsl7). These findings indicate that, by acting as curvature-sensing proteins, septins can localize signaling platforms in the cell. To test if septins can differentiate among micron-scale curvatures, Bridges et al. (2016) developed an elegant model system for septin assembly in vitro. They decorated silica beads with anionic phospholipid bilayers and measured the interaction affinity between purified fungal septin complexes and beads of different curvatures. Interestingly, septins were maximally recruited to “intermediate” sized beads (1.0–3.0 µm in diameter), with little to no recruitment to either very large (5.0–6.5 µm in diameter) or very small (0.3 µm in diameter) beads. These results indicate that septin filaments preferentially localize to a curvature (κ) of 0.7–2.0 µm⁻¹ in the absence of other cellular factors. To provide additional information on the mechanism of sensing, the authors purified mutant septin complexes that fail to polymerize into filaments and showed that the affinity of septins for micron-scale membrane curvature does not require filament formation per se. However, septins must polymerize into filaments for stable membrane association. Collectively, in vivo experiments using A. gossypii and in vitro experiments using silica beads highlight that septins have the intrinsic ability to recognize membrane curvature at the micron scale.Finally, to study the recognition of micron-scale membrane curvature beyond fungi, Bridges et al. (2016) turn their attention to human septins. Using tissue culture cells, they observe that the abundance of septins is associated with the degree of membrane curvature. To confirm these observations in vitro, they purified human septins and analyzed their binding affinity to silica beads with phospholipid bilayers. As seen with A. gossypii septins, human septins also showed a preference for beads ∼1.0 µm in diameter, strongly suggesting an evolutionarily conserved property of septins for sensing membrane curvature at the micron scale.Based on their findings, Bridges et al. (2016) propose that septins provide eukaryotic cells with a mechanism to recognize curvature at the micron scale. This feature differentiates septins from other sensor proteins that strictly detect curvature at the nanometer scale (e.g., BAR domain–containing proteins). However, it is likely that septins do more than recognize membrane, and the precise role of septins in membrane recognition remains unknown. The highly conserved structural and biochemical properties of septins suggest they organize, stabilize, and functionalize membrane domains (Caudron and Barral, 2009; Kusumi et al., 2012; Bridges and Gladfelter, 2015). Although we are far from knowing the full repertoire of septin function, this new work by Bridges et al. (2016) reminds us that understanding how membranes can specify septin assembly is essential to understand the role of septins in eukaryotic cells.     

Fifty years of cycling

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.

The cell cycle is a process that orders a number of cellular processes to ensure the accurate duplication of the cell. It was hoped that a genetic analysis would reveal how the events were integrated. The inspiration for this was the work of Bob Edgar and Bill Wood on bacteriophage morphogenesis, which revealed the ordered steps by which phage parts were assembled and then put together (Wood and Edgar, 1967). The major questions were how DNA replication and spindle morphogenesis were integrated to achieve accurate chromosome segregation; how cell division was integrated with mitosis to ensure that both daughter cells received a full chromosome complement; and how growth and division were integrated to maintain a constant cell size.Mutants that block cell cycle progression were identified by screening collections of randomly generated temperature sensitive mutants (Hartwell et al., 1970a). Each mutant was screened individually by time-lapse photomicroscopy to identify cell division control (CDC) mutants that caused all cells in the population to arrest at the same point in the cell cycle at the restrictive temperature. The use of budding yeast was critical because the presence and size of the daughter bud provided a simple readout of where cells were in the cell cycle. The first collection of CDC mutants was derived from screening 1500 temperature-sensitive mutants and identified a total of 147 mutants, which fell into 32 complementation groups (Hartwell et al., 1973). An example of one of the first mutants identified is shown in Figure 1. Wild-type cells are found at all stages of the cell cycle at the restrictive temperature (panel A), whereas the CDC mutant cells arrest in late in the cell cycle with large daughter buds (panel B).Open in a separate windowFIGURE 1:An example of one of the first CDC mutants isolated in budding yeast. Wild-type cells and temperature-sensitive mutant cells were grown at the permissive temperature and then shifted to the restrictive temperature, and CDCs were followed by photomicroscopy. (A) Wild-type cells, which are found at all stages of the cell cycle at the restrictive temperature, as indicated by the presence of cells at all stages of the daughter cell budding cycle. (B) A CDC mutant in which all cells have arrested at a cell cycle stage with large daughter buds.The phenotypes of the mutants revealed some preliminary answers to the major questions (Hartwell et al., 1970a, b). Assuming that the primary biochemical defect in a mutant was the process that stopped first, the following conclusions could be drawn. The elongation of the spindle was dependent on prior duplication of the spindle poles and the completion of DNA replication. Cell division and mitosis were coordinated because the formation of the daughter bud was dependent on spindle pole duplication in the previous cycle and cytokinesis was dependent on prior elongation of the spindle. Growth and division were coordinated because the CDC28 (CDK1) function at Start required sufficient growth to initiate all the events of the cell cycle. Cell fusion during mating of haploid cells was coordinated with cell division because mating hormones arrested the cell cycle at the CDC28 step and fusion was restricted to that step in the cell cycle.These observations raised the question of how the dependence of events on one another was controlled. Two models were considered. One, named substrate–product, proposed that a late step was dependent on an early step because the latter was the substrate for the former (e.g., replicated DNA was a substrate for the spindle). The other was regulation, meaning either that signals from completion of an early event induced a late event or that an incomplete early event inhibited a late event. In one example, regulation was evident when a genetic analysis of how damaged DNA arrested nuclear division revealed a signaling pathway (the DNA damage checkpoint) that also accounted for why incomplete DNA replication prevented mitosis (Weinert and Hartwell, 1988).The mechanisms underlying the dependence of cell cycle events upon one another have now been defined in considerable molecular detail. By way of illustration, we will briefly summarize what is known about the dependence of mitosis on replicated chromosomes and the dependence of cell division on mitosis. In some cases, the cdc mutants contributed to this work as a means to identify the relevant genes. However, in many cases, important components were not isolated as cdc mutants. Some of these have since been identified through biochemistry and subsequently shown to have Cdc phenotypes after mutants were created by in vitro mutagenesis. Additional cell cycle components were identified in other genetic screens or isolated using the original cdc mutants as starting points to search for genetic interactors. For example, the cyclins from yeast (which are redundant and nonessential), and even humans, were isolated, in part, as high-copy suppressors of the yeast cdc28 mutant (Hadwiger et al., 1989).One prominent class of cdc mutants affected DNA replication. Mutants targeting two of the three essential replicative polymerases were isolated, as were DNA ligase, a gene required for replication near telomeres, and genes required for the production of deoxyribonucleotides. We now know that these mutations resulted in robust arrest phenotypes because they led to the accumulation of significant amounts of ssDNA, the signal recognized by the replication checkpoint pathway (Zou and Elledge, 2003). This checkpoint signaling pathway both blocks mitosis and feeds back to replication. This feedback to replication helps stalled forks progress and also blocks origins that have not yet fired from doing so. The critical checkpoint phosphorylation events that effect these goals are well understood. Mitotic arrest is largely achieved by phosphorylation and stabilization of Pds1 (Cohen-Fix and Koshland, 1997), an event that blocks sister chromatid separation. Blocking origin firing is mediated by the phosphorylation of two proteins required for origin firing (Lopez-Mosqueda et al., 2010; Zegerman and Diffley, 2010). Finally, the restart of replication forks stalled by either mutational disruption or exogenous agents is promoted by the phosphorylation of several critical targets that increase nucleotide levels and modify the activity of proteins that act at the fork (Ciccia and Elledge, 2010). If measured by viability after fork arrest, this last function is by far the most significant role of this checkpoint pathway, although mitotic arrest and blocking origin firing are also important for preserving genome integrity.While the cdc screen was effective in identifying genes involved in the mechanics of DNA replication, it was less effective in identifying genes that function exclusively to establish origins of replication. An exception to this, CDC6, sheds some light on why this may be. cdc6 mutants brought to a fully nonpermissive temperature do not form replication forks, and thus do not activate the replication checkpoint, although they eventually arrest in mitosis due to the formation of an aberrant spindle that triggers the spindle assembly checkpoint (Piatti et al., 1995; Stern and Murray, 2001). Temperature-sensitive alleles of polymerase or ligases are likely to generate a few nonfunctional replication forks even when the alleles are weak, thus activating the checkpoint and providing a clear Cdc phenotype. Of course, it could not have been foreseen at the time of this screen that mutations in some cell cycle processes might eliminate the very signals for arrest that the screen was designed to identify.Major progress has also been made in understanding the coordination of events needed to ensure successful chromosome segregation to daughter cells during mitosis. The spindle poles duplicate and separate to form a microtubule-based spindle. During DNA replication, the cohesin complex is loaded onto sister chromatids to keep them paired until the metaphase to anaphase transition. At the same time, the kinetochores that mediate attachment of chromosomes to the spindle microtubules assemble on centromeres. These are examples of substrate–product relationships where cohesin and kinetochores will assemble once the chromatin templates are available. Similarly, kinetochores make attachments to the spindle microtubules as soon as they are assembled. The spindle assembly checkpoint, a regulatory pathway, monitors kinetochore–microtubule interactions and halts the metaphase-to-anaphase transition until all chromosomes are properly attached (Hoyt et al., 1991; Li and Murray, 1991). Once the checkpoint is satisfied, cells activate the anaphase-promoting complex to release the linkage between sister chromatids and allow the spindle to elongate and pull chromosomes to opposite poles. As the spindle elongates into the daughter cell, the cell reverses Cdc28 substrate phosphorylations to promote mitotic exit and cytokinesis.How are all of these events coordinated? Surprisingly, although corresponding temperature-sensitive mutants exist for most mitotic genes, the cdc screen did not isolate the structural components of the yeast spindle, spindle pole, or kinetochore, with the exception of one pole mutant, cdc31. In contrast, the screen identified many signaling molecules that regulate the metaphase-to-anaphase transition. The cdc screen identified five subunits of the anaphase-promoting complex, which coordinates this transition by ensuring the degradation of proteins that lead to the removal of cohesion from chromosomes and the down-regulation of Cdc28 activity (King et al., 1995; Sudakin et al., 1995). One of the substrates that must be degraded is Pds1, the same protein that is the target of the DNA checkpoint (Cohen-Fix et al., 1996). Pds1 inhibits the enzyme separase that releases cohesin to ensure the timely separation of sister chromatids (Ciosk et al., 1998; Uhlmann et al., 2000). The targeted degradation of Pds1 and cyclins by a single complex couples spindle elongation and chromosome segregation to Cdc28 inactivation. The spindle assembly checkpoint inhibits the anaphase-promoting complex, reinforcing the coordination between proper spindle attachment to chromosomes and anaphase progression (Hwang et al., 1998; Kim et al., 1998). After chromosome segregation, many Cdc28 substrates must also be dephosphorylated to exit from mitosis. Each of the essential kinases and phosphatases in this control system, called the mitotic exit network, were found in the cdc screen (Shou et al., 1999; Visintin et al., 1999). The mitotic exit network coordinates cytokinesis with the spindle delivering chromosomes to the daughter cell (Bardin et al., 2000; Pereira et al., 2000). Finally, several members of the septin ring that ensures cytokinesis, the last event in the cell cycle, were identified as cdc mutants. The septin mutants continue to bud, replicate DNA, and undergo mitosis in the next cell cycle, showing that completion of all events in the prior cell cycle is not necessarily required for progression. However, looking back, most of the key mitotic events are coordinated by regulatory events that reinforce the dependence of one event on the next, as opposed to the substrate–product relationship. Even in cases where there are clear substrate–product dependencies, such as spindle attachment to kinetochores, the cell has multiple regulatory mechanisms in place to halt the cell cycle until errors are detected and corrected, thus ensuring the proper execution of mitosis.What do the next 50 years hold? The short examples above illustrate the tremendous progress that has been made in understanding the molecular mechanisms that ensure the coordination of cell cycle events. However, there are still major questions about its specificity, accuracy, and complexity, as well as how it is altered in disease. Specialized cell divisions such as meiosis and asymmetric cell division or modified cell cycle states such as quiescence require modifications to the cell cycle. The reconstitution of molecular events has helped to identify the minimal components and regulation required, but this has not accounted for the exquisite precision of these processes in the cell. The complexity of how individual cell cycle events integrate with other cellular processes such as metabolism is still in the early stages. Uncontrolled cell division is the root of cancer, so identifying therapeutic targets that specifically cause cancer vulnerabilities and avoid toxicity to normal cell divisions is still very much needed. In sum, many of the principles gained from cell cycle research have guided our thinking about biological processes; further elucidating the underlying mechanisms of the cell cycle will continue to influence fundamental biology and disease research for decades to come.