UC San Diego

Living cells respond to environmental cues through complex signaling and gene regulatory networks. A common theme throughout this thesis will be exploring design principles in biological networks and how they operate dynamically to process information and make decisions.

In Chapter 1, we tackle how different types of stresses induce distinct nuclear translocation dynamics of Msn2, an outstanding question in the field. In the absence of stress, PKA phosphorylates Msn2, causing it to be exported out of the nucleus. In response to stress, PKA activity is inhibited, Msn2 is dephosphorylated and translocated into the nucleus. In response to glucose limitation, Msn2 exhibits an initial homogenous pulse of nuclear translocation followed by sporadic nuclear pulses with dose-dependent frequency, but in response to osmotic stress Msn2 undergoes a single translocation pulse with dose-dependent duration. We hypothesized that the difference between glucose limitation and osmotic stress-induced Msn2 dynamics might be a result of glucose limitation-dependent Snf1 activation, since previous studies suggest that Snf1 and PKA mutually inhibit each other. We use modeling and experiments to demonstrate that these different upstream network structures could, in fact, be responsible for the differences we see in Msn2 translocation dynamics.

In Chapter 2, we study a recurring scheme in gene regulatory networks, which is combinatorial gene regulation by seemingly redundant transcription factors (TFs), using time-lapse microscopy and microfluidics. We use the seemingly redundant yeast homologous stress responsive TFs Msn2 and Msn4 as a model to quantitatively study the functional relevance of closely related TFs in the same single cells and find that Msn2 and Msn4 have non-redundant and distinct functions in combinatorial gene regulation. In response to a transient input, either Msn2 or Msn4 alone is sufficient to induce the expression of target genes with fast kinetics promoters. Target genes with slow kinetics promoters, however, require activation of both Msn2 and Msn4 in these conditions. Importantly, slow kinetic promoter activation is dependent on duration of the upstream signal because in response to a prolonged input, slow kinetic promoter activation no longer requires both Msn2 and Msn4. Thus, in Chapter 2, we determine that coordinated gene regulation by seemingly redundant TFs is not fixed, but rather dependent on the dynamics of upstream signals.

In Chapter 3, we demonstrate that cells retain a memory of many of upstream signaling events that occur in response to stress, which primes the cells to respond to future severe stress events. We use microfluidics and time-lapse microscopy to modulate the amplitude and duration of priming stimulus and also increase the break time in between the priming stimulus and severe stress. Using this system, we have determined that cells acquire an amplitude-dependent short-term memory of priming stimulus, which is induced and lost rapidly, and a duration-dependent long-term memory which is stable for a long period of time before finally declining after 100 minutes. We use this information about the dynamical specificity of different types of cellular memory and their stability to determine the cellular pathways responsible for the observed memory.