Bud-Localization of CLB2 mRNA Can Constitute a Growth Rate Dependent Daughter Sizer.
ABSTRACT: Maintenance of cellular size is a fundamental systems level process that requires balancing of cell growth with proliferation. This is achieved via the cell division cycle, which is driven by the sequential accumulation and destruction of cyclins. The regulatory network around these cyclins, particularly in G1, has been interpreted as a size control network in budding yeast, and cell size as being decisive for the START transition. However, it is not clear why disruptions in the G1 network may lead to altered size rather than loss of size control, or why the S-G2-M duration also depends on nutrients. With a mathematical population model comprised of individually growing cells, we show that cyclin translation would suffice to explain the observed growth rate dependence of cell volume at START. Moreover, we assess the impact of the observed bud-localisation of the G2 cyclin CLB2 mRNA, and find that localised cyclin translation could provide an efficient mechanism for measuring the biosynthetic capacity in specific compartments: The mother in G1, and the growing bud in G2. Hence, iteration of the same principle can ensure that the mother cell is strong enough to grow a bud, and that the bud is strong enough for independent life. Cell sizes emerge in the model, which predicts that a single CDK-cyclin pair per growth phase suffices for size control in budding yeast, despite the necessity of the cell cycle network around the cyclins to integrate other cues. Size control seems to be exerted twice, where the G2/M control affects bud size through bud-localized translation of CLB2 mRNA, explaining the dependence of the S-G2-M duration on nutrients. Taken together, our findings suggest that cell size is an emergent rather than a regulatory property of the network linking growth and proliferation.
Project description:Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
Project description:The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation.
Project description:Two new B-type cyclin genes from Saccharomyces cerevisiae, called CLB5 and CLB6, are located in a tail to tail arrangement adjacent to the G2/M phase promoting cyclins CLB2 and CLB1, respectively. These genomic cyclin arrays are flanked by tRNAs and repeated sequences of Ty elements suggesting an intrachromosomal gene duplication followed by an interchromosomal gene duplication. Based on their deduced protein sequence the CLB5 and CLB6 genes form a new pair of B-type cyclins. They are most related to each other and then to the deduced protein sequence of their adjacent genes CLB1 and CLB2. Both genes are periodically expressed, peaking early in the cell cycle. Loss of function mutants are viable, but clb5- mutants exhibit a delay in S phase whereas clb6- mutants show a delay in late G1 and/or S phase. The clb5 mutant phenotype is somewhat more pronounced in a double null mutant. Both cyclins have the potential to interact with the p34CDC28 kinase in vivo.
Project description:The key molecular event that marks entry into the cell cycle is transcription of G1 cyclins, which bind and activate cyclin-dependent kinases. In yeast cells, initiation of G1 cyclin transcription is linked to achievement of a critical cell size, which contributes to cell-size homeostasis. The critical cell size is modulated by nutrients, such that cells growing in poor nutrients are smaller than cells growing in rich nutrients. Nutrient modulation of cell size does not work through known critical regulators of G1 cyclin transcription and is therefore thought to work through a distinct pathway. Here, we report that Rts1, a highly conserved regulatory subunit of protein phosphatase 2A (PP2A), is required for normal control of G1 cyclin transcription. Loss of Rts1 caused delayed initiation of bud growth and delayed and reduced accumulation of G1 cyclins. Expression of the G1 cyclin CLN2 from an inducible promoter rescued the delayed bud growth in rts1Delta cells, indicating that Rts1 acts at the level of transcription. Moreover, loss of Rts1 caused altered regulation of Swi6, a key component of the SBF transcription factor that controls G1 cyclin transcription. Epistasis analysis revealed that Rts1 does not work solely through several known critical upstream regulators of G1 cyclin transcription. Cells lacking Rts1 failed to undergo nutrient modulation of cell size. Together, these observations demonstrate that Rts1 is a key player in pathways that link nutrient availability, cell size, and G1 cyclin transcription. Since Rts1 is highly conserved, it may function in similar pathways in vertebrates.
Project description:In budding yeast four mitotic cyclins (Clb1-4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2?, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G 2/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.
Project description:Little is known about the pathways used by cyclins and cyclin-dependent kinases to induce the events of the cell cycle. In budding yeast, a protein called Nap1 binds to the mitotic cyclin Clb2, and Nap1 is required for the ability of Clb2 to induce specific mitotic events, but the role played by Nap1 is unclear. We have used genetic and biochemical approaches to identify additional proteins that function with Nap1 in the control of mitotic events. These approaches have both identified a protein kinase called Gin4 that is required for the ability of Clb2 and Nap1 to promote the switch from polar to isotropic bud growth that normally occurs during mitosis. Gin4 is also required for the ability of Clb2 and Nap1 to promote normal progression through mitosis. The Gin4 protein becomes phosphorylated as cells enter mitosis, resulting in the activation of Gin4 kinase activity, and the phosphorylation of Gin4 is dependent upon Nap1 and Clb2 in vivo. Affinity chromatography experiments demonstrate that Gin4 binds tightly to Nap1, indicating that the functions of these two proteins are closely tied within the cell. These results demonstrate that the activation of Gin4 is under the control of Clb2 and Nap1, and they provide an important step towards elucidating the molecular pathways that link cyclin-dependent kinases to the events they control.
Project description:Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Delta strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Delta cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Delta and hsl1Delta resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.
Project description:The mammalian cell cycle is governed by Cyclin-Dependent Kinases (CDKs) whose activities are restricted by the availability of their cyclins. Temporal control of cyclin levels, therefore, lies at the very core of cell-cycle regulation. While cyclins E, A, and B are well understood, the dynamic regulation of Cyclin D remains elusive. Using live-cell imaging and single-cell tracking, we report here that Cyclin D1 levels drop upon passage through the Restriction Point (R-point) in G1, stay low in early S phase, and rise again in late S and G2. We show that Cyclin D1 is continuously synthesized and degraded throughout the cell cycle, with degradation and synthesis rates varying substantially across cell-cycle phases. The G1 drop in Cyclin D1 levels is caused by a sudden increase in the degradation rate. Our findings thus fill a crucial gap in the core cell-cycle control network.
Project description:B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.
Project description:Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.