A single-cell study on stochasticity growth and gene expression

Life of single cells is not deterministic, but virtually all processes in a cell are subject to stochastic fluctuations. As consequence, genetically identical cells, which are living in the same environment, can behave differently. They can, for example, produce different amounts of proteins, grow at different rates and can even specialize into completely different phenotypes. The discovery of cell-to-cell variability opened up many questions, ranging from what are the origins of the fluctuations, to whether its consequences are detrimental or beneficial for cells.
The aim of this thesis is to better understand stochasticity in gene expression and growth rate of bacterial cells. To this end, we investigate whether, next to gene expression, also growth rate of single cells fluctuates dynamically, which we will show to be the case. We then focus on the following questions about gene expression and growth: What are the origins of fluctuations? Can fluctuations propagate from one to the other? To address these questions, we work with a model organism, the bacteriumE. coli . This organism is simple enough to allow for quantitative experiments, but already so complex that many processes inside these cells have not been understood yet.
Studying stochastic fluctuations in gene expression and growth rate in single cells requires precise and rather high throughput tools to measure these quantities. In chapter 2 and 3 we describe these methods. By using automated time-lapse microscopy, we acquire movies of growing E. coli cells which are expressing fluorescent proteins. We then use automated analysis software to segment, track and analyze cells. To accurately measure gene expression, a fluorescent protein reporter of high quality is needed. Therefore, we present a comparison of different fluorescent proteins which assesses their suitability for time-lapse experiments.
In chapter 4 we investigate the general interdependence of fluctuations in gene expression and growth rate. We find that the noise intensities of both fluctuations are very strongly correlated and that they scale linearly. In apparent contrast to that, the fluctuating time traces are shown to be only modestly correlated. We develop a linear noise model to explain these observations and analyze what constraints the linear scaling imposes on such models. A central result is that the intensities of different cellular noise sources do not change independently, but are set by one single global parameter.
In chapter 5 we study the effects of a specific source of fluctuations in gene expression: the bacterial cell cycle. As cells grow and prepare for division, they copy their chromosome. With two copies of each gene and an increasing cell size, the production rate of proteins increases. We show that about half of the noise in protein production rate is caused by gene duplication. In contrast to that, protein concentration is hardly affected by the cell cycle because the exponential volume increase almost perfectly cancels the increase in protein production rate.
The fact that gene expression fluctuates has been known for many years. However, it was unclear whether such fluctuations also affect the growth rate of cells. This question is addressed in chapter 6. We show that fluctuations in enzymes can indeed propagate and cause growth fluctuations. Conversely, growth fluctuations also propagate back to disturb protein concentration. We develop an analytical model to accurately predict noise transmission. Our results indicate that fluctuations can not only propagate through gene networks but also through metabolic reactions. They also suggest that cellular metabolism is inherently stochastic.
Finally, in chapter 7 we investigate the influence of fluctuations in ribosome concentration on the growth rate. Because ribosomes are the molecular machines responsible for protein production, they play a central role in cellular growth. Each cell needs thousands of ribosomes and as producing these large numbers is costly, the ribosome production is tightly regulated and adjusted to the need. Still, their concentration in single cells fluctuates, and we here investigate whether these fluctuations affect growth rate. Our data suggests that fluctuations in ribosome concentration do not propagate to growth, which indicates that ribosomes are not dynamically limiting growth rate.