The challenges in finding your home as a multidisciplinary scientist.

Technological advances in a variety of scientific disciplines are being applied in the life sciences leading to an increase in the number scientists who see themselves or are classed as being multidisciplinary. Although their diverse skills are celebrated and needed to understand the immense complexity of life, being a multidisciplinary researcher can pose unique challenges. We asked multidisciplinary researchers and the director of an institute that fosters multidisciplinary research for their thoughts on what they see as the challenges or obstacles that multidisciplinary scientists can often face.

CDK Substrate Phosphorylation and Ordering the Cell Cycle.

S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.

Sizing up to divide: mitotic cell-size control in fission yeast.

Schizosaccharomyces pombe is a good model to study cell-size control. These cells integrate size information into cell cycle controls at both the G1/S and G2/M transitions, although the primary control operates at the entry into mitosis. At G2/M there is both a size threshold, demonstrated by the fact that cells divide when they reach 14 µm in length, and also correction around this threshold, evident from the narrow distribution of sizes within a population. This latter property is referred to as size homeostasis. It has been argued that a population of cells accumulating mass in a linear fashion will have size homeostasis in the absence of size control, if cycle time is controlled by a fixed timer. Because fission yeast cells do not grow in a simple linear fashion, they require a size-sensing mechanism. However, current models do not fully describe all aspects of this control, especially the coordination of cell size with ploidy.

TOR regulates variability of protein synthesis rates.

Cellular processes are subject to inherent variability, but the extent to which cells can regulate this variability has received little investigation. Here, we explore the characteristics of the rate of cellular protein synthesis in single cells of the eukaryote fission yeast. Strikingly, this rate is highly variable despite protein synthesis being dependent on hundreds of reactions which might be expected to average out at the overall cellular level. The rate is variable over short time scales, and exhibits homoeostatic behaviour at the population level. Cells can regulate the level of variability through processes involving the TOR pathway, suggesting there is an optimal level of variability conferring a selective advantage. While this could be an example of bet-hedging, but we propose an alternative explanation: regulated 'loose' control of complex processes of overall cellular metabolism such as protein synthesis, may lead to this variability. This could ensure cells are fluid in control and agile in response to changing conditions, and may constitute a novel organisational principle of complex metabolic cellular systems.

Core control principles of the eukaryotic cell cycle.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

The cell cycle and cell size influence the rates of global cellular translation and transcription in fission yeast.

How the production of biomass is controlled as cells increase in size and proceed through the cell cycle events is important for understanding the regulation of global cellular growth. This has been studied for decades but has not yielded consistent results, probably due to perturbations induced by the synchronisation methods used in most previous studies. To avoid this problem, we have developed a system to analyse unperturbed exponentially growing populations of fission yeast cells. We generated thousands of fixed single-cell measurements of cell size, cell cycle stage and the levels of global cellular translation and transcription. We show that translation scales with size, and additionally, increases at late S-phase/early G2 and early in mitosis and decreases later in mitosis, suggesting that cell cycle controls are also operative over global cellular translation. Transcription increases with both size and the amount of DNA, suggesting that the level of transcription of a cell may be the result of a dynamic equilibrium between the number of RNA polymerases associating and disassociating from DNA.

The cell in an era of systems biology.

The increasing use of high-throughput technologies and computational modeling is revealing new levels of biological function and organization. How are these features of systems biology influencing our view of the cell?

The TOR-dependent phosphoproteome and regulation of cellular protein synthesis.

Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR-dependent growth inhibition. Here, we present a resource providing an in-depth characterisation of Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR-dependent phospho-regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR-regulated targets both in fission yeast and other eukaryotes.

Cell division intersects with cell geometry.

Single-celled organisms monitor cell geometry and use this information to control cell division. Such geometry-sensing mechanisms control both the decision to enter into cell division and the physical orientation of the chromosome segregation machinery, suggesting that signals controlling cell division may be linked to the mechanisms that ensure proper chromosome segregation.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle.

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

A CDK activity buffer ensures mitotic completion.

The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion.

Establishing the program of origin firing during S phase in fission Yeast.

Initiation of eukaryotic DNA synthesis occurs at origins of replication that are utilized with characteristic times and frequencies during S phase. We have investigated origin usage by evaluating the kinetics of replication factor binding in fission yeast and show that similar to metazoa, ORC binding is periodic during the cell cycle, increasing during mitosis and peaking at M/G1. At an origin, the timing of ORC binding in M and pre-RC assembly in G1 correlates with the timing of firing during S, and the level of pre-IC formation reflects origin efficiency. Extending mitosis allows ORC to become more equally associated with origins and leads to genome-wide changes in origin usage, while overproduction of pre-IC factors increases replication of both efficient and inefficient origins. We propose that differential recruitment of ORC to origins during mitosis followed by competition among origins for limiting replication factors establishes the timing and efficiency of origin firing.

Identification of mutants with increased variation in cell size at onset of mitosis in fission yeast.

Fission yeast cells divide at a similar cell length with little variation about the mean. This is thought to be the result of a control mechanism that senses size and corrects for any deviations by advancing or delaying onset of mitosis. Gene deletions that advance cells into mitosis at a smaller size or delay cells entering mitosis have led to the identification of genes potentially involved in this mechanism. However, the molecular basis of this control is still not understood. In this work, we have screened for genes that when deleted increase the variability in size of dividing cells. The strongest candidate identified in this screen was mga2 The mga2 deletion strain shows a greater variation in cell length at division, with a coefficient of variation (CV) of 15-24%, while the wild-type strain has a CV of 5-8%. Furthermore, unlike wild-type cells, the mga2 deletion cells are unable to correct cell size deviations within one cell cycle. We show that the mga2 gene genetically interacts with nem1 and influences the nuclear membrane and the nuclear-cytoplasmic transport of CDK regulators.

Science on the streets of the Big Apple.

A five-day festival of science takes place this week at venues across New York City. The festival features not only leading researchers from New York and beyond but also actors, writers, musicians, and choreographers in a series of multimedia programs designed to reveal science to the general public in exciting new ways.

Replication origin selection regulates the distribution of meiotic recombination.

The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology.