Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biochemistry & Biophysics
Non Technical Summary
Knowing which cellular pathways, and how these pathways, affect the machinery of cell division will allow modulations of cell proliferation. Once cells initiate their division, they are committed to completing it. Hence, what determines when cells initiate their division also dictates how fast cells multiply. However, it is virtually unknown which cellular pathways affect initiation of division, which factors operate within each pathway and the extent of interactions between pathways. We propose experiments to answer these questions in baker's yeast. This model organism is suited for genetic and biochemical studies.?Completion of the proposed work will provide a much clearer picture of the molecular events that trigger cells to divide than it is available. The studies we propose are significant because they focus on a group of genes that has largely unexplored roles in cell cycle progression, influencing the field considerably. This project will enable in the future precise control of initiation of cell division and cell proliferation.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Problem Statement Most cells must grow before they can divide, but how do cells determine when they have grown enough so they can commit to a new round of cell division? In the past decades, an impressive body of research identified the machinery that initiates cell division in eukaryotes. However, even in well-studied microbial systems, we do not know how nutrient, metabolic or other "growth" inputs activate this machinery. It is as if we have before us an intricate engine, with little knowledge about what turns it on. Without such knowledge, one can neither predict, nor modulate the metabolic control of cell division at will. Objectives Our long-term goal is to predict when cells will begin dividing, with inputs of our choice. Here, we will define how growth pathways in response to diverse inputs activate the cell division machinery. Do growth needs for cell division reflect hierarchical or distinct, separable pathways? Our central hypothesis is that growth requirements for cell division are multiple, distinct and separable. We reached this view through systematic screens of factors that control cell division in budding yeast [1, 2]. Several variables affect the timing of initiation of division: cell size at birth, the size cells have to reach when they commit to division, and how fast they reach that size [1, 2]. We found that mutants in metabolic enzymes or ribosomal parts differ in these variables. Some mutants affect the size at birth, size at initiation of division, the rate of growth, or any combination of the above. Hence, with regard to when cells initiate division, mutants in growth pathways occupy a large, unexplored phenotypic space. The rationale of this proposal is that our findings enable us to query that phenotypic space. We will define the growth pathways that trigger division in response to nutrients, which factors function in each pathway, the interactions among pathways, and how each pathway is molecularly linked to cell division. We integrate genetic, molecular and engineering methods to complete these objectives: 1. Build a functional network among components of central metabolism, ribosomes, TOR signaling and cell division. We will quantify the epistatic relationships of all binary mutants, scoring variables that report accurately on the timing of initiation of cell division. 2. Position diverse nutrient inputs relative to pathways that trigger cell division. The growth rate of steady-state microbial cultures in chemostats is set externally. This allows nutrient-specific effects on cell division to be studied separately from their effects on growth rate. However, standard chemostats are low throughput, complex and costly. We developed a microfluidic chemostat array that overcomes these obstacles [3]. With this technology, we will query how different nutrients engage growth pathways to control division. 3. Identify targets of translational control that link protein synthesis with initiation of division. Metabolism underpins the protein synthesis capacity of the cell, but what are the translational targets that are relevant for initiation of division? We identified ribosomal mutants with distinct effects on cell division. We will use these mutants to pinpoint mRNAs with altered translation. We will then test the corresponding gene products for their role in cell division. This project is transformative and original. The proposed work builds on new concepts, methods and technologies we developed: We defined and expanded the variables that report accurately when cells initiate division [1, 2]. We devised the approaches to measure these variables [1, 2]. We constructed prototype microfluidic devices that enable us to probe efficiently the nutrient-mediated control of cell division [3].
Project Methods
1. The system: We use the budding yeast S. cerevisiae to delineate the fundamental links between cellular growth and division. This organism has several properties that make it ideal for our objectives: i) Initiation of DNA replication is coupled to the formation of a bud. Thus, one can monitor the timing of initiation of division by phase microscopy. ii) It is possible to obtain minimally perturbed, homogeneous, synchronous cell populations. iii) It can grow in steady-state continuous cultures. This allows for precise control and monitoring of metabolic parameters. iv) S. cerevisiae is a genetically tractable eukaryote, with the most developed tools available, including various comprehensive strain and plasmid collections. Collectively, these properties are unattainable in any other system. 2. Querying the timing of initiation of cell division: In proliferating cells, the G1 phase of any given cell cycle lasts from the end of the previous mitosis until the beginning of DNA synthesis. In unfavorable growth conditions, eukaryotic cells typically stay longer in G1, delaying initiation of DNA replication [12-16]. Subsequent cell cycle transitions, culminating with mitosis, are less sensitive to growth limitations, and their timing does not vary greatly, even if growth conditions worsen. Thus, differences in the length of G1 account for most of the differences in total cell cycle, or generation times, between the same cells growing in different media. The lack of a detailed view of upstream regulatory networks that govern the timing of initiation of cell division in S. cerevisiae is surprising, given the rich history of the field. How has the problem been approached over the years? Decades ago, a relationship between the size or mass of a cell and the timing of initiation of DNA replication was shown from bacterial [17], to mammalian cells [18]. Indeed, a newborn budding yeast cell is smaller than its mother is, and it will not initiate cell division until it becomes bigger [12, 13]. Thus, it appears that there is a size threshold for initiation of division in yeast. Based on this concept of a critical size, the question of "when do cells divide?" was reduced to "what size are cells when they divide?" Hence, several screens for regulators of initiation of cell division interrogated cell size [19-23]. Systematic, genome-wide approaches to find such regulators relied solely on cell size changes [19, 20]. Over the years, the molecular circuitry that comprises the switch that needs to be activated for initiation of cell division has been revealed [24-27]. Sadly, however, in any one of influential reviews of the last 20 years (e.g., [28-30]), vague terms such as "cell size" or "growth cues" describe how that switch is activated, without any molecular basis of what these terms mean. To examine cell cycle progression more directly, we used flow cytometry to measure the DNA content of all S. cerevisiae non-essential gene deletions [1]. Contrary to expectations, we reported very little correlation between the DNA content of mutant strains and the mean cell size of these mutants [1]. Subsequent work by others confirmed these observations [31]. These findings force a major reevaluation of the role of size homeostasis in cell division. Overall, the emphasis on critical size mutants to identify mechanisms that determine the timing of initiation of cell division is problematic for two reasons: First, it could lead to errors about the actual timing of initiation of division; Second, it does not allow the sampling of gene products that do not affect size homeostasis. A prime example of the latter is cells lacking TOR1, which encodes a Target of Rapamycin kinase. The key growth signaling role of Tor1p is well established [32], yet the critical size of tor1? cells is normal. Such a phenotype is typical for most gene products required for the correct timing of initiation of cell division [1, 2], including gene products with roles in metabolism or other "growth" pathways, such as protein synthesis. Using size as a proxy for "growth" is convenient and reasonable. However, focusing only at the critical size and ignoring other variables introduces very serious bias in the analysis. Instead, we reasoned that variations of G1 length among different mutants, or growth in different nutrients, could arise from differences in the boundaries of G1 in each case (e.g., different mutants may enter and/or exit G1 at different sizes) and differences in the rate at which cells traverse G1 in each case. A small size at the time of initiation of division would lead to an accelerated initiation of division, but only if it is not accompanied by any other changes that collectively prolong G1. This is the case for some mutants with truly accelerated initiation of division, such as whi5D [33, 34]. However, as we have argued in the literature [1, 2], a small size at division in some other mutants (e.g., in sfp1? cells, lacking a regulator of ribosome biogenesis) was not sufficient for acceleration of division. Their small critical size notwithstanding, we concluded that sfp1? cells severely delay initiation of cell division because they are born small and grow slowly [1]. Ignoring other parameters that also affect the overall length of the G1 phase, such as birth size and growth rate, could lead to erroneous conclusions about the timing of initiation of division. Unfortunately, birth size values are rarely measured, or incorporated, in estimates of G1 progression. To address this lack of information, we developed computational approaches that allowed us to present the first genome-wide, birth size dataset [2]. From highly synchronous elutriated cultures, we can also determine the specific rate of size increase and critical size [1], allowing us to measure the length of G1 accurately. We argue that analyzing the mutants we identified with the new, unbiased and inclusive approaches we have developed to monitor G1 progression from birth to the time cells initiate a new round of cell division will allow us to delineate the growth pathways that determine when cells divide.