Biology subjects

Schloop, A. E.

Publications and source records attributed to Schloop, A. E..

3 recordsLinked to original sources

bioRxiv2024

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

The NF-{kappa}B 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-{kappa}B and its inhibitor I{kappa}B, which sequesters NF-{kappa}B to the cytoplasm of cells until needed. It is also known that I{kappa}B can enter nuclei, disrupt NF-{kappa}B binding to DNA, and shuttle it out to again sequester NF-{kappa}B in the cytoplasm. In Drosophila melanogaster, a homologous system between the proteins Dorsal (homologous to NF-{kappa}B) and Cactus (homologous to I{kappa}B) 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-{kappa}B pathway in early Drosophila embryos and highlights the power of advanced optical techniques for investigating complex biological dynamics.

developmental biology↗

bioRxiv2020

Discovery of membrane-permeating cyclic peptides via mRNA display

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

biochemistry↗

bioRxiv2019

Shuttling of Dorsal by Cactus: mechanism and implications

In 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-{kappa}B 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/I{kappa}B facilitates the diffusion of Dorsal/NF-{kappa}B. 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.

developmental biology↗