A reaction-diffusion network model predicts a dual role of Cactus/IκB to regulate Dorsal/NFκB nuclear translocation in Drosophila

Barros, Claudio D. T.; Cardoso, Maira Arruda; Bisch, Paulo Mascarello; Araujo, Helena; Lopes, Francisco J. P.

doi:10.1371/journal.pcbi.1009040

Cited by 3 publications

(2 citation statements)

References 66 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This study has provided general insights into the time of action of morphogens and suggests that the regulation of nuclear import-export kinetics might be important in transcription factor dynamics, providing support for recently generated models (Barros et al 2021;Schloop, Bandodkar, and Reeves 2020). These data also suggest a general principle of how gene expression might respond to the dynamics of morphogens in addition to sensing their concentration.…”

Section: Discussionsupporting

confidence: 67%

Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

McGehee,

Stathopoulos

2024

Preprint

View full text Add to dashboard Cite

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that result in either nuclear export or degradation of Dorsal when exposed to blue light. Nuclear export of Dorsal results in loss of expression for the high threshold, ventrally-expressed target gene snail (sna) but retention of the low threshold, laterally-expressed target gene short-gastrulation (sog). In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels than when Dorsal is exported from the nucleus. To elucidate how nuclear export results in loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found it reenters the nucleus even under conditions of blue-light when export is favored. The associated kinetics of being imported and exported continuously are likely responsible for loss of sna but, alternatively, are able to support sog. Collectively, our results show that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention.

show abstract

Section: Discussionsupporting

confidence: 67%

Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

McGehee,

Stathopoulos

2024

Preprint

View full text Add to dashboard Cite

show abstract

“…3D). These estimates of Cact biophysical parameters can help constrain predictive models of the Dorsal gradient (Ambrosi et al, 2014; Barros et al, 2021; Carrell et al, 2017; Kanodia et al, 2009; O’Connell & Reeves, 2015).…”

Section: Resultsmentioning

confidence: 99%

Spatiotemporal dynamics of NF-κB/Dorsal inhibitor IκBα/Cactus inDrosophilablastoderm embryos

Schloop,

Hiremath,

Shaikh

et al. 2024

Preprint

View full text Add to dashboard Cite

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. InDrosophila 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 vivovisualization 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 GFPin 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 inDrosophilaembryos 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 dynamicsin 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 earlyDrosophilaembryos and highlights the power of advanced optical techniques for investigating complex biological dynamics.

show abstract

Dorsal/NF-κB exhibits a dorsal-to-ventral mobility gradient in the Drosophila embryo

Asafen

Yousef

2018

Preprint

View full text Add to dashboard Cite

Morphogen-mediated patterning is a highly dynamic developmental process. To obtain an accurate understanding of morphogen gradients, biophysical parameters such as protein diffusivities must be quantified in vivo. The dorsal-ventral (DV) patterning of early Drosophila embryos by the NF-κB homolog Dorsal (Dl) is an excellent system for understanding morphogen gradient formation. Dl gradient formation is controlled by the inhibitor Cactus/IκB (Cact), which regulates the nuclear import and diffusion of Dl protein. However, quantitative measurements of spatiotemporal Dl movement are currently lacking. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of Dl. We find that the diffusivity of Dl varies along the DV axis, with lowest diffusivities on the ventral side, and the DV asymmetry in diffusivity is exclusive to the nuclei. Moreover, we also observe that nuclear export rates are lower in the ventral and lateral regions of the embryo. Both cross correlation spectroscopy measurements and a computational model of Dl/DNA binding suggest that DNA binding of Dl, which is more prevalent on the ventral side of the embryo, is correlated to a lower diffusivity and nuclear export rate. We propose that the variation in Dl/DNA binding along the DV axis is dependent on Cact binding Dl, which prevents Dl from binding DNA in dorsal and lateral regions of the embryo. Thus, our results highlight the complexity of morphogen gradient dynamics and the need for quantitative measurements of biophysical interactions in such systems. Significance StatementTissues in developing organisms are patterned through signaling mechanisms that utilize morphogen concentration gradients. To better understand how these gradients form and are maintained, biophysical parameters such as protein diffusivities must be quantified in vivo. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of the morphogen Dorsal (Dl) in the Drosophila embryo. We find that both the diffusivity and nuclear export rate of Dl varies along the dorsal-to-ventral axis, with the lowest values on the ventral side. Moreover, incorporating these experimentally determined diffusion coefficients into a mathematical model suggests that DNA binding of Dl correlates with lower Dl movement. Thus, our results highlight how quantitative measurements of biophysical parameters can improve our understanding of morphogen gradient dynamics.

show abstract

A reaction-diffusion network model predicts a dual role of Cactus/IκB to regulate Dorsal/NFκB nuclear translocation in Drosophila

Cited by 3 publications

References 66 publications

Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation

Spatiotemporal dynamics of NF-κB/Dorsal inhibitor IκBα/Cactus inDrosophilablastoderm embryos

Dorsal/NF-κB exhibits a dorsal-to-ventral mobility gradient in the Drosophila embryo

Contact Info

Product

Resources

About