Spatiotemporal dynamics of NF-κB/Dorsal inhibitor IκBα/Cactus in Drosophila blastoderm embryos

The NF-κB signaling pathway is a key regulatory network in mammals that controls many cellular processes, including immunity and inflammation. Of particular note is the relationship between NF-κB and its inhibitor IκBα, which sequesters NF-κB to the cytoplasm of cells until needed. It is also known that IκBα can enter nuclei, disrupt NF-κB binding to DNA, and shuttle it out to again sequester NF-κB in the cytoplasm. In Drosophila melanogaster , a homologous system between the proteins Dorsal (homologous to NF-κB) and Cactus (homologous to IκBα) is important in embryo development, specifically in establishment of the Dorsal nuclear concentration gradient. Previous work suggests Cactus also enters the nucleus; mathematical models of the Dorsal gradient fail to accurately predict the normal range of the gradient without nuclear Cactus. However, direct, in vivo visualization of Cactus spatiotemporal dynamics, including its localization to the nuclei, has been difficult to gather. Previously, imaging Cactus in live embryos was complicated by rapid protein turnover, preventing fluorescent protein fusions from fully maturing. To address this, we used the CRISPR/Cas9 system to tag Cactus with the recently developed “LlamaTag” (LT), a genetically encodable nanobody from llamas that dynamically binds to GFP in vivo . We then employed standard confocal imaging, as well as advanced optical techniques such as raster image correlation spectroscopy (RICS) and fluorescent recovery after photobleaching (FRAP) to investigate the spatiotemporal distribution of Cactus-LlamaTag in Drosophila embryos at the blastoderm stage. Our results demonstrate that Cactus can be found in the nuclei of early embryos, consistent with its role as a transcription factor regulator. Moreover, by using the data from FRAP and RICS, we were able to estimate biophysical parameters of Cactus dynamics in vivo , including its nuclear transport rate constants and fraction bound to GFP. These data were further used to constrain a mathematical model that allowed us to infer experimentally inaccessible biophysical parameters, such as the concentration of Cact protein and the dissociation constant of LT and GFP. Our study provides new insights into the regulation of the NF-κB pathway in early Drosophila embryos and highlights the power of advanced optical techniques for investigating complex biological dynamics.