Frederick Cross, Ph.D. - US grants

The focus of our work is the regulation of the G1/S transition in the budding yeast, S. cerevisiae. Previous work in several labs, including ours, has established the CLN1, CLN2, and CLN3 genes of yeast as critical in regulation of this transition, both by external factors (hormones, nutrients) and internal factors (cell size). The homology of these genes with cyclins, and their genetic and biochemical interactions with the product of the cdc2 homolog in this organism, CDC28, combined with the apparent specificity of their action to the G1/S transition (as opposed to the G2/M transition for previously described cyclins) suggests the idea that the CLN proteins may be 'G1 cyclins'. Since the G1/S transition is a primary point of regulation in many organisms, including humans, and since the regulation of this transition is grossly abnormal in cancer, it is important to determine the mechanism of action and regulation of CLN genes, as well as to determine the universality through evolution of the system. These are the issues addressed in this application.

Budding yeast mate in the Gl phase of the cell cycle. Cells of each mating type produce mating pheromones that interact with cell surface receptors on cells of the opposite mating type. Binding of pheromone to receptors triggers a differentiative program including altered gene transcription, altered cell morphology, and Gl cell cycle arrest. The START regulatory point in the Gl phase of the cell cycle marks a sharp transition from sensitivity to mating pheromones to resistance until the next Gl phase. We are interested in the molecular basis of this commitment event, which integrates conjugation and the cell cycle. The FAR1 gene is essential for mating pheromone arrest. We have found that FAR1 degradation is cell-cycle regulated, and degradation correlates with phosphorylation. This control and FAR1 transcriptional control result in significant accumulation of Far1 protein only in the pre-START Gl phase of the cell cycle. Cell cycle regulation of FAR1 thus can contribute to the transition from mating pheromone sensitivity to resistance at START. This implies a negative control by cell-cycle progression on machinery involved in cell cycle arrest and mating. We have begun genetic and biochemical analysis of the basis for the control of Far1 degradation. This analysis is based on our observations that Far1 phosphorylation precedes its degradation, and that an N-terminal deletion mutant of Far1 blocks degradation. We have identified an additional independent negative control of the overall pheromone signalling pathway. Transcriptional induction by mating factor of genes involved in mating is largely blocked at about the time of START, possibly by specific Cln/Cdc28 protein kinase complexes. We wish to characterize this control, first genetically and ultimately biochemically.

proteins; plants; enzymes; biomedical resource;

Integration of the Cell Cycle with Signal Transduction from the Mating Factor Pathway. This signal transduction pathway inhibits G I cyclin function. G I cyclins CLN1 and CLN2 (but not CLN3 or B-type cyclins, including CLB5) have the reciprocal activity of inactivating the mating factor pathway. We are investigating the mechanism of this reciprocal regulation, which is likely due to regulation of the PAK-related protein kinase Ste20. Mass Spectrometry is being used to map and monitor the phosphorylation of Ste20 and mutants of Ste20 under a variety of conditions

The ongoing accumulation of vast collections of DNA sequence data has catalyzed the development of novel approaches for profiling the expression of genes at the mRNA level. These methods, while extraordinarily powerful, do not provide direct information on changes either in the levels of proteins or their states of modifications. The development of analogous high throughput methods for directly monitoring protein levels, while increasingly desirable for biological investigations in the post-genome era, presents a formidable analytical challenge. Although recent advances in the use of mass spectrometry (MS) in conjunction with protein/DNA-sequence database search-algorithms allows for the identification of proteins with unprecedented speed, it remains difficult to obtain accurate quantitative information concerning the levels of the identified proteins and the levels of site-specific modifications within individual protein molecules. In the absence of appropriate antibodies, quantitation is usually achieved by autoradiography after metabolic radiolabeling, fluorography, or the use of protein stains. These procedures are dependent on complete separation of the proteins of interest by techniques such as high-resolution 2-D electrophoresis. There remains a pressing need for easier, more reliable means to rapidly profile protein levels. We have devised a novel general method for accurately comparing levels of individual proteins present in cell pools that differ in some respect from one another (e.g., the presence of a mutated gene) and for accuractely determining changes in the levels of modifications (e.g., phosphorylation) at specific sites on the individual proteins. The procedure can be applied to mixtures of proteins, obviating the need for complete separation.

Recently, evidence was presented that the Cln2-Cdc28 cyclin-dependent kinase inhibits the mating factor signal transduction pathway by interfering with the function of Ste20, and that this inhibition correlates with Cln2-dependent in vivo phosphorylation of Ste20. We used MALDI-TOF-MS and LC-ESI-ion trap-MS/MS analysis of unlabeled full length Ste20 as well as a truncated form spanning residues 496-939 (Ste20trunc) to identify 13 sites that were phosphorylated in vivo. Here, we apply the our newly developed method for quantitating changes in the level of phosphorylation to specifically identify Cln2-dependent in vivo phosphorylation sites in Ste20. We use the data to test the hypothesis that Cln2-dependent phosphorylation of Ste20 brings about inhibition of the mating factor transduction pathway.

Although much progress has been made in determining the mechanisms of regulation of cyclin/cyclin dependent kinase activation, little is known about substrates for these protein kinase complexes. Still, it is considered likely that cyclins contribute to substrate selectivity for the different complexes, resulting in the specificity of e.g. Cln2/Cdc28 for G1/S events vs. that of Clb2/Cdc28 for G2/M events (note that the Cdc28 protein kinase catalytic subunit is the same in both cases). In order to examine this idea critically, and in order to begin to elucidate the mechanisms of action of cyclin-dependent kinases in triggering diverse cell cycle transitions, it is necessary to identify and characterize phosphorylation substrates of these complexes, to determine if any or all are specifically phosphorylated by individual cyclin/Cdc28 complexes, and to determine the functional consequences of these phosphorylation events for cell cycle control. We are designing exp eriments t hat will utilize our newly developed MS approaches to assist in the identification of these potentially important phosphorylation substrates.

Cyclin-dependent kinases (Cdk's) are critical in regulation of the cell cycle in all eukaryotes. Cdk's are stringently regulated by cyclin binding, and cyclin availability thus is one of the main means by which cell cycle progression is regulated. This proposal is for experiments to examine the consequences of deregulation of this system. In one set of experiments, a strategy is proposed for mutational activation of the budding yeast Cdk Cdc28 independent of the cyclin binding requirement. Achievement of this goal (towards which significant progress has been made) will allow dissection of the roles of cyclins in simple enzyme activation as opposed to targeting of the enzyme to specific substrates. In a second set of experiments, we propose examining the consequences of making the level and activity of a particular B-type cyclin/Cdc28 complex, Clb5-Cdc28, constitutive through the cell cycle. Although current models predict that this should halt the cell cycle in G1 phase due to interference with function of origins of DNA replication, our results suggest that this may not be the case. We propose to characterize control of Clb5 degradation through the cell cycle, and the role of the Clb5 destruction box in its degradation. We propose experiments to test the hypothesis that Clb5 is intrinsically specialized for S phase entry while another B-type cyclin, Clb2, is intrinsically specialized for entry into mitosis. This hypothesis is in contrast to current models in which all B-type cyclins are equally capable of both positive and negative regulation of DNA replication.

Cyclin-dependent kinases are critical for eukaryotic cell cycle control. In budding yeast, 9 different cyclins all activate the same cyclin- dependent kinase Cdc28, resulting in diverse biological responses depending on the cyclin complexed to Cdc28. Little is known about the basis for cyclin specificity of action. The G1 cyclins of budding yeast, CLN1, CLN2 and CLN3, are required for the Start transition at the beginning of the cell cycle, and act by binding to and activation the Cdc28 cyclin-dependent kinase catalytic subunit. Although the yeast G1 cyclins are functionally redundant, they nevertheless differ in their biological mode of action. This system may provide a model for cyclin specificity of action in eukaryotic in eukaryotic cell cycle control. This question is significant for human health because of the involvement of multiple human G1 cyclins in controlling cell proliferation. This proposal comprises direct examination of the contrasting effects of different cyclins on subcellular localization of the cyclin-Cdk complex, and analysis of a highly cyclin-specific biological response, the regulation of the mating factor response pathway by the G1 cyclin Cln2.

This renewal application is to continue work on how cyclins drive the cell cycle. The role of multi-site phosphorylation of the G1 stabilizers Sic1 and Cdh1. The G1 period of the cell cycle is refractory to B-type cyclin dependent kinase because of accumulation of the Sid stoichiometric inhibitor, and because of highly active B-type cyclin proteolysis due to Cdh1. Multi-site phosphorylation of both Sic1 and Cdh1 by G1 cyclins have been considered essential for exit from G1. We have found, though, that expression of unphosphorylatable Sic1 (all 9 Cdk sites mutated) from the endogenous locus results in a fully viable strain, though with a lengthened G1. We will similarly test the properties of unphosphorylatable Cdh1 expressed from the endogenous promoter. These experiments will address the dynamic consequences of multisite phosphorylation of G1 regulators by cyclin-Cdk complexes, at physiological levels. Interactions of B-type cyclins with cell cycle execution machinery. While a lot is known about the cell cycle oscillator controlling levels of cyclin-dependent kinase and anaphase-promoting complex, much less is known about how these activities eventually drive the actual events of the cell cycle such as DMAreplication or spindle function. Our recent results indicate that the high degree of redundancy of the six yeast B-type cyclin genes is only apparent: non-essential 'checkpoint'surveillance mechanisms and other regulatory safeguards become essential in the absence of cyclin-specific pathways. Disabling these regulatory safeguards allows focus on cell biological pathways controlled by specific cyclins. Cdc14: targets and regulators. Cdc14 is a phosphatase required for exit from mitosis;it is released from sequestration in the nucleolus just before mitotic exit. Cdc14 probably dephosphorylates Cdk targets and thus helps reverse the mitotic state, but it is unresolved if Cdc14 is specific in vivo for a few critical targets, or alternatively dephosphorylates most or all Cdk substrates. We have exploited mutants affecting Cdc14 localization to explore the spectrum of Cdc14 targets. In additional studies we will determine the consequences of blocking Pds1 degradation, at endogenous expression levels, and the functional significance of Cdk-mediated phosphorylation of the mitotic exit kinase Dbf2. These experiments will probe the dynamic consequences of Clb kinase-Cdc14 phosphatase antagonism in cell cycle regulation. Overall, we are interested in regulation of cell cycle dynamics, and in cyclin-specific pathways promoting individual cell cycle events.

mass spectrometry

Antibodies; CRISP; Cell Cycle; Cell Cycle Progression; Cell Division Cycle; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Housing; Institution; Investigators; Localized; Methods; NIH; National Institutes of Health; National Institutes of Health (U.S.); Phosphatases; Phosphohydrolases; Phosphomonoesterases; Phosphoric Monoester Hydrolases; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; Yeasts; gene product; magnetic beads; mutant

DESCRIPTION (provided by applicant): Single-cell variation (noise) in the operation of genetic networks is an inevitable consequence of the small size of cellular systems, leading to small numbers of molecules (genes, mRNAs, proteins) that may cause fluctuations in the function of important genetic control circuits. Where such noise is deleterious, it is likely that noise suppression mechanisms have evolved;noise could also be beneficial in some cases, by allowing breakout from dynamical dead-ends or by diversifying a population to meet a changing environment. Despite the significance of this topic, empirical results on noise have so far been largely restricted to simple synthetic gene circuits. We propose analysis of single-cell variation in a much more complex and physiologically relevant situation: the robustness of cell cycle traversal and duration of cell cycle intervals in the eukaryote S. cerevisiae. We have developed a quantitative fully automated time- lapse fluorescent microscopy setup allowing semi-automated analysis of cell-cycle-regulated gene expression and relocation of cell cycle regulators, throughout the generation of complete multi-cell cycle pedigrees. This capability can be integrated with the enormous amount of information available about the regulation of the budding yeast cell cycle, the availability of genetic reagents to systematically perturb the cell cycle, and useful deterministic mathematical models of the cell cycle engine. We will test the hypothesis that variability in G1 length is due to bistability at cell cycle Start. We will generate quantitative single-cell data on the relationship of cell and nuclear size to cell cycle transition times, in wild-type and mutants, to provide constraints for models on nucleo-cytoplasmic size coupling. We will attack the question of what promoter architectures may lead to different levels of single-cell variability in gene expression, using a synthetic promoter strategy combined with single-cell imaging. We have obtained direct evidence for stochastic molecular variation in cell cycle Start;we will pursue this finding genetically and with stochastic modeling. To pursue these ideas in more dynamic depth, we have developed microfluidic methods that allow periodic pulses of gene expression in individual monitored cells as they proliferate from single cells into microcolonies. With this technology we will test the response of the cell cycle engine to brief forced expression of regulators, and looking for mode-locking and other informative dynamic behaviors. We will extend these analytical tools to mitotic regulation, testing the hypothesis that the complex design of the mitotic oscillator functions to suppress noise. The results should shed light on fundamental questions of robustness and dynamics of the cell cycle engine at single-cell resolution, and on the advantages and disadvantages of variability in the operation of a fundamental complex genetic circuit.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1 which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A) which allows binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared to the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events. A paper describing this work is in press: J. Bloom, I.M. Cristea, A. Procko, V. Lubkov, B.T. Chait, M. Snyder, F. R. Cross Global analysis of CDC14 phosphatase reveals diverse roles in mitotic processes JBC In press

Cell cycle control is highly conserved through the eukaryotic kingdom, and the budding yeast model system has been the source of major insights applicable to issues in human development and disease. This proposal continues systems-level analysis of the eukaryotic cell cycle using this model system. The cell cycle 'clock' functions with high reliability and low noise, even though individual components and circuits making up the clock are frequently known to be highly variable. For example, gene expression is known to be highly variable between individual cells, and yet cell-cycle-regulated gene expression can be highly reliable with respect to timing and amplitude. Threshold responses to rising cyclin-Cdk activity levels can provide switch-like behavior, but such switches can frequently come at the cost of highly variable onset time; the overall cell cycle control circuitry avoids this variability. We are pursuing an emerging concept of multiple independent oscillators contributing to cell cycle control; while uncoupled oscillators result in highly variable and irregular sequences of cell cycle events, we propose that coupling ('phase-locking') of otherwise independent oscillators to the central cyclin-Cdk oscillator can yield a robust and accurate overall system. This proposal continues our innovative use of quantitative time-lapse fluorescence microscopy, over multi-cell cycle timescales, combined with semi-automated image analysis and in-depth genetic and quantitative analysis to drive systems-level understanding of cell cycle control. We are developing new methods of mathematical modeling. There is a pressing need in biology for simple but experimentally constrained models that can reveal basic control principles. The challenge is to find the most illuminating balance between the detail required for a connection to biological reality, and model simplicity required for transparency and insight. We are exploring methods to use geometrical, low-dimensionality representations of the cell cycle control network that can still be experimentally constrained, and that will yield testable predictions. In a new direction to provide evolutionary contrast from a critical but underexplored branch of the eukaryotic kingdom, we will carry out a genetic screen aiming at saturated detection of cell cycle control elements in the green alga, Chlamydomonas reinhardtii. We have devised robotic methods for the microbiology of mutant isolation, which combined with deep sequencing, allows a massive speedup of this project compared to traditional means.

Project summary Cell cycle control in yeast and animals (`Opisthokonts') is well understood, in broad principles as well as specific conserved mechanisms. However, in eukaryotic evolution, yeasts are more closely related to animals than to other eukaryotic kingdoms. Therefore, insights from Opisthokonts might apply poorly to earlier- diverging branches such as the plant kingdom, which is absolutely essential to life on earth. It is of great significance to understand cell cycle control in non-Opisthokont eukaryotes, both to understand these important kingdoms, and to elucidate the evolution of cell cycle control from the last eukaryotic common ancestor, illuminating what features of cell cycle control are truly ancestral and fundamental. Here I propose use of a microbial `plant', the green alga Chlamydomonas reinhardtii, to carry out a broad-spectrum genetic screen to molecularly identify cell cycle control genes. We will use these mutants to carry out functional analysis to determine similarities and differences from the Opisthokont paradigm. There are two reasons for studying Chlamydomonas in this context: (1) it's much more closely related to land plants than other microbial models; (2) it provides a model organism to study ancient pathways that were lost in fungal lineages due to rapid evolution, such as cilia, the G1/S control network (cyclin D, Rb, E2F/DP). To attack this problem, we have developed an efficient pipeline for isolation, identification and analysis of conditional mutations in cell cycle control genes. The procedures integrate classical genetics with robotics, next-generation sequencing and novel bioinformatics approaches for rapid and efficient molecular identification of hundreds of essential genes (~150 identified to date, with many more in the pipeline). Mutant screens are prerequisite for in-depth analysis of specific biological pathways, to provide a well- populated `parts' list and initial functional classification based on simple phenotypic assays. As the project progresses, in addition to aiming for a comprehensive cell cycle collection, we are focusing on specific genes and pathways, with priority to those that are plant-kingdom-specific. We will examine the cyclin-Cdk-APC control system, where our results in Chlamydomonas and previous results in land plants indicate substantial conservation but also significant divergence from the yeast/animal model. In addition, we will collaborate to characterize mechanisms of Chlamydomonas cytokinesis. Cytokinesis outside of yeast/animals proceeds without an actomyosin contractile ring. We have evidence from characterizing mutants already obtained for the role of actin and actin-interacting components. For both these aims, we will tag key proteins with fluorescent epitopes to allow subcellular localization in time-lapse microscopy, in wild type and appropriate mutant backgrounds, and analyze regulation of protein abundance and function through the cell cycle. Clusters of mutants have already revealed essential pathways that will be further studied, including pathways controlling cell cycle commitment, mitotic progression and cytokinesis.