BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. I will discuss our work using a combination of single molecule and super resolution microscopy methods. We found that BAX and BAK present distinct oligomerization properties, and that BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. We visualized how BAX and BAK regulate each other as they co-assemble into the same apoptotic pores. Our results indicate that the relative availability of BAX and BAK molecules determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.

The past two years of the Covid pandemic have made mathematical modeling of viral infections broadly visible to the general public. Models have been used to predict future infection numbers as much as number of patients in intensive care and on ventilators, with results being presented in TV talk shows as well as in executive government meetings and parliament discussions. It is fair to say that mathematical models have had a significant impact on political decisions and on the course of the pandemic in the past 2 years. Interestingly, there is no strong scientific community on epidemiological modeling in Germany. Most models have been developed by groups that are rooted in different fields. I will talk about about the journey I have taken, from developing mathematical models of viral replication and immune response at the level of individual cells at BioQuant, their extension to patient and drug treatment data, to recent models that we have developed in the context of the Covid-19 pandemic.

Cells respond to external stimuli and developmental cues mostly with changes in gene expression. Gene expression is often regulated by transcription factors (TFs), proteins that bind DNA in a sequence-specific manner at so-called response elements and recruit the general transcription machinery at the promoter to start transcription. TFs have been shown to display different dynamics, that is, the shape of the curve that describes the TF activity over time is not always the same, but depends on the type of stimulus the cell receives. Different dynamics have been, in turn, implicated in the activation of specific sets of target genes. In light of these findings, it is proposed that the same TF starts different gene expression programs purely depending on its dynamics and not its total abundance. This would imply that promoters be able to decode TF dynamics. In this lecture, I will present our work combining optogenetic experiments, automated image analysis and mathematical modeling to understand which elements on a mammalian promoter play a role in decoding TF dynamics. I will first describe LINuS, a light-inducible nuclear localization system developed by us, then show how, by imposing various TF dynamics with blue light and reading out the response from a library of synthetic promoters built with well-studied and defined elements, we find that sustained and pulsatile activation are distinguishable provided the coupling between TF binding and transcription pre-initiation complex formation is inefficient. Additionally, we show that the efficiency of translation initiation affects the ability of the promoter to sense TF dynamics. Using the knowledge acquired, we built a synthetic circuit that allows us to obtain two gene expression programs (proteins A and B both highly expressed versus protein A highly expressed and protein B only very weakly expressed) depending solely on TF dynamics. Taken together, these results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build synthetic circuits that respond better to determined TF dynamics.

Advanced fluorescence microscopies including but not limited to super-resolution techniques have seen tremendous progress over the past 15 years. While in the early days the developments were mainly driven by improvements of the optical microscopes and associated procedures for data acquisition, more recent developments with protein tags and click chemistry have opened new opportunities for improving and creating novel fluorescent probes significantly contributing to the advancements in fluorescence microscopy. In this context, we have explored the use of reversible and irreversible chemical reactions to control the emissive and none-emissive states of fluorescent probes. This not only enabled alternative approaches for multiplexing but also resulted in novel probes for single molecule localization microscopy (SMLM) and correlative microscopy approaches in living cells. Thorough studies of the involved photo physical process ultimately led to a new class of fluorescent probes that can be used in live-cell experiments without the need of further washing procedures.