Bistable switches that produce all-or-none responses have been found to regulate a number of natural cellular decision making processes, and subsequently synthetic switches were designed to exploit their potential. However, an increasing number of studies, particularly in the context of cellular differentiation, highlight the existence of a mixed state that can be explained by tristable switches. The criterion for designing robust tristable switches still remains to be understood from the perspective of network topology. To address such a question, we calculated the robustness of several 2- and 3-component network motifs, connected via only two positive feedback loops, in generating tristable signal response curves. By calculating the effective potential landscape and following its modifications with the bifurcation parameter, we constructed one-parameter bifurcation diagrams of these models in a high-throughput manner for a large combinations of parameters. We report here that introduction of a self-activatory positive feedback loop, directly or indirectly, into a mutual inhibition loop leads to generating the most robust tristable response. The high-throughput approach of our method further allowed us to determine the robustness of four types of tristable responses that originate from the relative locations of four bifurcation points. Using the method, we also analyzed the role of additional mutual inhibition loops in stabilizing the mixed state.
Keywords: bifurcation; cell fate; network motifs; potential landscape; robustness; tristability.