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

2018 ◽  
Author(s):  
Hadel Al Asafen ◽  
Natalie M. Clark ◽  
Thomas Jacobsen ◽  
Rosangela Sozzani ◽  
Gregory T. Reeves
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Developmental Process ◽  
Ventral Side ◽  
Correlation Spectroscopy ◽  
Drosophila Embryo ◽  
Morphogen Gradient ◽  
Biophysical Parameters ◽  
Quantitative Measurements ◽  
Gradient Formation

AbstractMorphogen-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.

scholarly journals Shuttling of Dorsal by Cactus: mechanism and implications

2019 ◽  
Author(s):  
Allison E. Schloop ◽  
Sophia Carrell-Noel ◽  
Gregory T. Reeves
Keyword(s):  
Cell Types ◽  
Bmp Signaling ◽  
Previous Literature ◽  
Drosophila Embryo ◽  
Morphogen Gradient ◽  
Facilitated Diffusion ◽  
Morphogen Gradients ◽  
Binding Partner ◽  
Early Drosophila Embryo ◽  
Gradient Formation

AbstractIn a developing animal, morphogen gradients act to pattern tissues into distinct domains of cell types. However, despite their prevalence in development, morphogen gradient formation is a matter of debate. In our recent publication, we showed that the Dorsal/NF-κB morphogen gradient, which patterns the DV axis of the early Drosophila embryo, is partially established by a mechanism of facilitated diffusion. This mechanism, also known as “shuttling,” occurs when a binding partner of the morphogen facilitates the diffusion of the morphogen, allowing it to accumulate at a given site. In this case, the inhibitor Cactus/IκB facilitates the diffusion of Dorsal/NF-κB. In the fly embryo, we used computation and experiment to not only show that shuttling occurs in the embryo, but also that it enables the viability of embryos that inherit only one copy of dorsal maternally. Here we further discuss our evidence behind the shuttling mechanism, the previous literature data explained by the mechanism, and how it may also be critical for robustness of development. Finally, we describe an interaction between Dorsal and BMP signaling that is likely affected by shuttling.

scholarly journals Cyto-architecture constrains the spread of photoactivated tubulin in the syncytial Drosophila embryo

2020 ◽  
Vol 64 (4-5-6) ◽  
pp. 275-287
Author(s):  
Sameer Thukral ◽  
Bivash Kaity ◽  
Bipasha Dey ◽  
Swati Sharma ◽  
Amitabha Nandi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Division ◽  
The Body ◽  
Drosophila Embryo ◽  
Morphogen Gradient ◽  
Cytoplasmic Domains ◽  
Microtubule Network ◽  
Drosophila Embryogenesis ◽  
Gradient Formation ◽  
Kinetics Of

Drosophila embryogenesis begins with nuclear division in a common cytoplasm forming a syncytial cell. Morphogen gradient molecules spread across nucleo-cytoplasmic domains to pattern the body axis of the syncytial embryo. The diffusion of molecules across the syncytial nucleo-cytoplasmic domains is potentially constrained by association with the components of cellular architecture. However, the extent of restriction has not been examined. Here we use photoactivation (PA) to generate a source of cytoplasmic or cytoskeletal molecules in order to monitor the kinetics of their spread in the syncytial Drosophila embryo. Photoactivated PA-GFP and PA-GFP-Tubulin generated within a fixed anterior area diffused along the antero-posterior axis. These molecules were enriched in the cortical cytoplasm above the yolk-filled center, suggesting that the cortical cytoplasm is phase separated from the yolk-filled center. The length scales of diffusion were extracted using exponential fits under steady state assumptions. PA-GFP spread a greater distance as compared to PA-GFP-Tubulin. Both molecules were more restricted when generated in the center of the embryo. The length scale of spread for PA-GFP-Tubulin increased in mutant embryos containing short plasma membrane furrows and a disrupted tubulin cytoskeleton. PA-GFP spread was unaffected by cyto-architecture perturbation. Taken together, these data show that PA-GFP-Tubulin spread is restricted by its incorporation in the microtubule network and intact plasma membrane furrows. This photoactivation based analysis of protein spread allows for interpretation of the dependence of gradient formation on syncytial cyto-architecture.

scholarly journals Cyto-architecture constrains a photoactivation induced tubulin gradient in the syncytial Drosophila embryo

2019 ◽  
Author(s):  
Sameer Thukral ◽  
Bivash Kaity ◽  
Bipasha Dey ◽  
Swati Sharma ◽  
Amitabha Nandi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Division ◽  
The Body ◽  
Drosophila Embryo ◽  
Morphogen Gradient ◽  
Cytoplasmic Domains ◽  
Microtubule Network ◽  
Cellular Architecture ◽  
Gradient Formation ◽  
Kinetics Of

AbstractDrosophila embryogenesis begins with nuclear division in a common cytoplasm forming a syncytial cell. Morphogen gradient molecules spread across nucleo-cytoplasmic domains to pattern the body axis of the syncytial embryo. The diffusion of molecules across the syncytial nucleo-cytoplasmic domains is potentially constrained by association with the components of cellular architecture, however the extent of restriction has not been examined so far. Here we use photoactivation (PA) to generate a source of cytoplasmic or cytoskeletal molecules in order to monitor the kinetics of their spread in the syncytial Drosophila embryo. Photoactivated PA-GFP and PA-GFP-Tubulin within a fixed anterior area diffused along the antero-posterior axis. These molecules were enriched in cortical cytoplasm above the yolk-filled center suggesting that the cortical cytoplasm is phase separated from the yolk-filled center. The length scales of diffusion were extracted using exponential fits under steady state assumptions. PA-GFP spread to greater distance as compared to PA-GFP-Tubulin. Both gradients were steeper and more restricted when generated in the center of the embryo probably due to a higher density of nucleo-cytoplasmic domains. The length scale of diffusion for PA-GFP-Tubulin gradient increased in mutant embryos containing short plasma membrane furrows and disrupted tubulin cytoskeleton. The PA-GFP gradient shape was unaffected by cyto-architecture perturbation. Taken together, these data show that PA-GFP-Tubulin gradient is largely restricted by its incorporation in the microtubule network and intact plasma membrane furrows. This photoactivation based analysis of protein spread across allows for interpretation of the dependence of gradient formation on the syncytial cyto-architecture.

scholarly journals Extracellular interactions and ligand degradation shape the nodal morphogen gradient

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yin Wang ◽  
Xi Wang ◽  
Thorsten Wohland ◽  
Karuna Sampath
Keyword(s):  
Correlation Spectroscopy ◽  
Cell Surface Receptor ◽  
Morphogen Gradient ◽  
Axis Formation ◽  
Cellular Interactions ◽  
Fluorescence Correlation ◽  
Selective Ligand ◽  
Surface Receptor ◽  
First Time

The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes. Nodal morphogens play critical roles in embryonic axis formation in many organisms. Models proposed to generate the Nodal gradient include diffusivity, ligand processing, and a temporal activation window. But how the Nodal morphogen gradient forms in vivo remains unclear. Here, we have measured in vivo for the first time, the binding affinity of Nodal ligands to their major cell surface receptor, Acvr2b, and to the Nodal inhibitor, Lefty, by fluorescence cross-correlation spectroscopy. We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy. We also investigated the contribution of ligand degradation to the Nodal gradient. We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range. By computational simulations of gradient formation, we demonstrate that diffusivity, extra-cellular interactions, and selective ligand destruction collectively shape the Nodal morphogen gradient.

p65 controls NF-κB activity by regulating cellular localization of IκBβ

2011 ◽  
Vol 434 (2) ◽  
pp. 253-263 ◽  
Author(s):  
Taras Valovka ◽  
Michael O. Hottiger
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cellular Processes ◽  
Dna Binding Activity ◽  
Localization Signal ◽  
Export Signal

NF-κB (nuclear factor κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by IκB (inhibitory κB) proteins which control nuclear–cytoplasmic shuttling and DNA binding of NF-κB. In the present paper, we describe a novel role for p65 as a critical regulator of the cellular localization and functions of NF-κB and its inhibitor IκBβ. In genetically modified p65−/− cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the NLS (nuclear localization signal) and the NES (nuclear export signal) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to the nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65-specific NLS and NES. The results of our present study also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA-binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB–IκBβ complexes. Taken together, the findings of the present study indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway.

scholarly journals Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

2008 ◽  
Vol 313 (1) ◽  
pp. 408-419 ◽  
Author(s):  
Takuya Akiyama ◽  
Keisuke Kamimura ◽  
Cyndy Firkus ◽  
Satomi Takeo ◽  
Osamu Shimmi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Morphogen Gradient ◽  
Gradient Formation
Sign in / Sign up

Export Citation Format

Share Document