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Volume is a fundamental morphological feature of cells, influencing a wide variety of cellular processes. Because it is coupled to so many processes, cells employ regulatory mechanisms to ensure cells exhibit a limited range of sizes. Recent work in budding yeast has shown that a key cell cycle regulator, the G1/S transcriptional inhibitor Whi5, is synthesized independent of cell size. The dilution of Whi5 in larger cells links cell size to G1/S cell cycle progression. However, it has also been shown that growth over the full cell cycle does not depend on cell size at birth, termed an "adder". It has been proposed that this observation suggests that cell size is controlled over the course of the full cell cycle, leading to an apparent contradiction. Here we show that cell size control occurs independently in different parts of the cell cycle and does not reflect a molecular mechanism measuring growth during the full cell cycle. Consistent with previous results, we find that cell size sets the rate of entry into the cell cycle during the pre-Start period. We also identify the key parameters predicting the rate of entry into cytokinesis at the end of the post-Start period. We use these parameters to build a phenomenological cell cycle model that recapitulates observations of growth and size distributions for cells without explicit coupling between cell cycle phases. Our model predicts that changes to the rate of progression through either phase of the cell cycle should disrupt the adder behavior and we show that mutants in genes controlling G1/S size control breaks the adder. The rate of passage through Start depends on volume, which is thought to depend on the size-independent expression of Whi5. This type of gene expression scaling is unusual because although cells of a given type may span a range of sizes, most proteins and RNA are maintained at constant, size independent, concentrations, rather than amounts. This ensures that biochemical reactions proceed independently of cell size. The identification of WHI5, whose gene product differs from this pattern, raises two fundamental questions: (1) Are there additional genes whose synthesis is decoupled from cell volume? (2) If most gene expression is proportional to cell size, what molecular mechanism promotes cell-size-independent gene expression? To address these questions, we analyzed flow cytometry data collected using the yeast GFP-fusion library. We identified approximately 200 genes whose expression is not proportional to cell volume. Gene ontology analysis revealed that non-scaling genes are enriched for genes with roles in DNA-templated processes and membrane transport. This suggests that cells employ differential protein synthesis to coordinate protein requirements with the scaling properties of cellular structures. Membranes are expected to scale as size2/3 and DNA content is independent of size. To understand the mechanisms that underlie size-independent gene expression, we used transcriptional reporters of non-scaling genes, including WHI5, and determined that cell-size-independent regulation of some genes is due to non-scaling transcription rates. Targeted analysis of the WHI5 promoter showed that the region between 1000 bases and 550 bases upstream of the translation start site are required for cell-size-independent gene expression. This suggests there is a molecular element within this region required for non-scaling gene expression. Finally, we identify a partitioning mechanism ensuring proteins are partitioned in dividing cells in amounts that are independent of asymmetric sizes of the mother and daughter cells. Tight chromatin association ensures that proteins are segregated in equal amounts despite asymmetric division. Consistent with this model, while Whi5 is normally partitioned in equal amounts, a Whi5 protein that lacks the domain required for association with transcription factors is partitioned in proportion to the mother-daughter cell size ratio. Taken together, our work demonstrates a functional role for differential size-dependency of protein synthesis and gives insights into the underlying molecular mechanism(s).
Polyploidy, increased copy number of whole chromosome sets in the genome, is a common cellular state in evolution, development and disease. Polyploidy enlarges cell size and alters gene expression, producing novel phenotypes and functions. Although many polyploid cell types have been discovered, it is not clear how polyploidy changes physiology. Specifically, whether the enlarged cell size of polyploids causes differential gene regulation has not been investigated. In this thesis, I present the evidence for a size-sensing mechanism that alters gene expression in yeast. My results indicate a causal relationship between cell size and gene expression. Ploidy-associated changes in the transcriptome therefore reflect transcriptional adjustment to a larger cell size. The causal and regulatory connection between cell size and transcription suggests that the physical features of a cell (such as size and shape) are a systematic factor in gene regulation. In addition, cell size homeostasis may have a critical function - maintenance of transcriptional homeostasis.
The first part of this thesis address a question formulated more than 80 years ago (and still remains elusive): how does a cell control its size? Growth of a cell and its subsequent division into daughters is a fundamental aspect of all cellular living systems. During these processes, how do individual cells correct size aberrations so that they do not grow abnormally large or small? How do cells ensure that the concentration of essential gene products are maintained at desired levels, in spite of dynamic/stochastic changes in cell size during growth and division? ☐ In chapter 1, we introduce the reader to the field of cell size/content homeostasis. We review how advances in singe-cell technologies and measurements are providing unique insights into these questions across organisms from prokaryotes to human cells. More specifically, how diverse strategies based on timing of cell-cycle events, regulating growth, and number of daughters are employed to maintain cell size homeostasis. We further discuss how size-dependent expression or gene-replication timing can buffer concentration of a gene product from cell-to-cell size variations within a population. ☐ In chapter 2, we propose the use of stochastic hybrid systems as a framework for studying cell size homeostasis. We assume that cell grows exponentially in size (volume) over time and probabilistic division events are triggered at discrete time intervals. We first consider a scenario, where a timer (i.e., cell-cycle clock) that measures the time since the last division event regulates both the cellular growth and division rates. We also study size-dependent growth / division rate regulation mechanisms. We provide bounds on different statistical indicators (mean, variance, skewness, etc). Additionally, we assess the effect of different physiological parameters (growth rate, partition errors, etc) on cell size distribution. ☐ Chapter 3 introduces a mechanistic model that might explain the recently uncovered added principle, i.e., selected species add a fixed size (volume) from birth to division, irrespective of their size at birth. To explain this principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. We show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This fact is corroborated through experimental data available. The model also suggest that the distribution of the added volume when scaled by its mean become invariant of the growth rate, a fact also veri ed through available experimental data. ☐ In part 2 of this thesis, we study which strategies are implemented by a viral species, ranging from bacteriophages to human immunodeficiency virus (HIV), in order to exploit host resources. In chapter 4, we review the classical theory of viral-host dynamics and describe the key knobs that viruses tweak to exploit a cell population. This theory suggest that viruses might evolved to have infinite infectivity and virulence. In the case of infectivity, chapter 5 gives an alternative to infinite infectivity: virus will evolve to moderate infectivity because of local interactions. As an example, we study a phage attacking a bacterial population. We include the effect of local interactions by assuming that the phage needs to scape from bacterial death remains (debris). ☐ Infinite virulence is also challenged as evolutionary alternative for viral propagation. In chapter 5 we study environments where availability of susceptible bacteria fluctuates across time. Under such scenarios bacteria behaves contrary to classical ecology theory: phages evolve to a moderate virulence (lysis time). We present this insights through the use of the stochastic hybrid system framework. ☐ In chapter 7, we present a mathematical model of HIV transmission including cell-free and cell-cell transmission pathways. A variation of this model is considered including two populations of virus. The first infects cells only by the cell-free virus pathway, and the second infects cells by either the cell-free or the cell-cell pathway (synapse-forming virus). Synapse-forming HIV is shown to provide an evolutionary advantage relative to non synapse-forming virus when the average number of virus transmitted across a synapse is a su ciently small fraction of the burst size. ☐ HIV disease is well-controlled by the use of combination antiviral therapy (cART), but lifelong adherence to the prescribed drug regimens is necessary to prevent viral rebound and treatment failure. Populations of quiescently infected cells form a "latent pool" which causes rapid recurrence of viremia whenever antiviral treatment is interrupted. A "cure" for HIV will require a method by which this latent pool may be eradicated. Current efforts are focused on the development of drugs that force the quiescent cells to become active. Previous research has shown that cell-fate decisions leading to latency are heavily in uenced by the concentration of the viral protein Tat. While Tat does not cause quiescent cells to become active, in high concentrations it prevents a newly infected cell from becoming quiescent. In chapter 8, we introduce a model of the effects of two drugs on the latent pool in a patient on background suppressive therapy. The first drug is a quiescent pool stimulator, which acts by causing quiescent cells to become active. The second is a Tat analog, which acts by preventing the creation of new quiescently infected cells. We apply optimal control techniques to explore which combination therapies are optimal for different parameter values of the model.
In recent years, the study of the plant cell cycle has become of major interest, not only to scientists working on cell division sensu strictu , but also to scientists dealing with plant hormones, development and environmental effects on growth. The book The Plant Cell Cycle is a very timely contribution to this exploding field. Outstanding contributors reviewed, not only knowledge on the most important classes of cell cycle regulators, but also summarized the various processes in which cell cycle control plays a pivotal role. The central role of the cell cycle makes this book an absolute must for plant molecular biologists.
Calcium Entry Channels in Non-Excitable Cells focuses on methods of investigating the structure and function of non-voltage gated calcium channels. Each chapter presents important discoveries in calcium entry pathways, specifically dealing with the molecular identification of store-operated calcium channels which were reviewed by earlier volumes in the Methods in Signal Transduction series. Crystallographic and pharmacological approaches to the study of calcium channels of epithelial cells are also discussed. Calcium ion is a messenger in most cell types. Whereas voltage gated calcium channels have been studied extensively, the non-voltage gated calcium entry channel genes have only been identified relatively recently. The book will fill this important niche.
Recent breakthroughs in the field of cell growth, particularly in the control of cell size, are reviewed by experts in the three major divisions of the field: growth of individual cells, growth of organs, and regulation of cell growth in the contexts of development and cell division. This book is an introductory overview of the field and should be adaptable as a textbook.
A much-needed guide through the overwhelming amount of literature in the field. Comprehensive and detailed, this book combines background information with the most recentinsights. It introduces current concepts, emphasizing the transcriptional control of genetic information. Moreover, it links data on the structure of regulatory proteins with basic cellular processes. Both advanced students and experts will find answers to such intriguing questions as: - How are programs of specific gene repertoires activated and controlled? - Which genes drive and control morphogenesis? - Which genes govern tissue-specific tasks? - How do hormones control gene expression in coordinating the activities of different tissues? An abundant number of clearly presented glossary terms facilitates understanding of the biological background. Speacial feature: over 2200 (!) literature references.
Growth and division are the two most important processes in plant organogenesis. Cell size results from the dynamic combination of these two processes. Hence studying cell size patterning is crucial to understand organogenesis. The Arabidopsis sepal epidermis is an ideal system to study cell size as it forms a characteristic cell size pattern ranging from cells with only one hundredth the length of the sepal (small cells) to cells with one-fifth the length of the sepal (giant cells). Small cells are produced by ordinary mitosis whereas giant cells are produced by endoreduplication. In my dissertation, I addressed how cell size patterning is generated by genetic and genomic approaches. In my first study, I discovered that the endomembrane trafficking protein SEC24A suppresses endoreduplication in an ACR4, DEK1 and LGO dependent manner. SEC24A, the first identified giant cell formation inhibitor, unraveled a hidden layer of the complicated regulatory network of cell size patterning. In my second study, I applied translating ribosome affinity purification coupled with deep sequencing (TRAP-seq) to systematically discover changes in gene expression between differently sized cells. It was discovered that the giant and small cells have very distinct translatomes, implying they are different cell types. In addition, by comparing the giant cell and another highly endoreduplicated cell type, the trichome, we discovered that although endoreduplication triggers common downstream responses, the giant cell and trichome do have distinct genomic regulation modules. Cell biology and genetics were applied and validated the high-throughput sequencing results.