Angesichts der Fülle von Beiträgen, die sich mit Theodor Fontanes Leben und Werk befassen, ist es erstaunlich, dass zur Landpartie bisher noch keine eigenständige Untersuchung vorliegt. Diese Studie ...widmet sich dem Thema erstmals umfassend und fokussiert es mit Blick auf sozial-, kultur- und medienhistorische Kontexte ebenso wie auf literatur- und kunsthistorische Perspektiven immer wieder neu. Leitend ist dabei die These, dass die Landpartie im Romanwerk Fontanes einen Topos konstituiert, der stets dieselben Ordnungs- und Strukturprinzipien aufweist. Dieses Erzählmuster wird in den einzelnen Texten allerdings sehr produktiv und variantenreich gestaltet. Kern der narrativen Gestaltung ist dabei stets eine Verknüpfung von Liebes- und Geselligkeitshandlung. Dies verleiht der Landpartie handlungsdynamisierendes Potential, weshalb ihr eine wichtige konzeptionelle Funktion für die Romane Fontanes zukommt.
The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, ...we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing.
For complete details on the use and execution of this protocol, please refer to Aguilar et al.1
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•Guide for in-frame knockin design, protein tag selection, and locus analysis•Step-by-step protocol for the cloning of gRNAs and SEED donors•Instructions for injecting Drosophila embryos•Guidance on screening, harvesting, and confirmation of knockins
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing.
Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. We address Notch gene ...regulation using
as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). Here, we generate a novel
allele by genome engineering. Phenotypically,
behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, we define the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the
background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations.
Angesichts der Fulle von Beitragen, die sich mit Theodor Fontanes Leben und Werk befassen, ist es erstaunlich, dass zur Landpartie bisher noch keine eigenstandige Untersuchung vorliegt. Diese Studie ...widmet sich dem Thema erstmals umfassend und fokussiert es mit Blick auf sozial-, kultur- und medienhistorische Kontexte ebenso wie auf literatur- und kunsthistorische Perspektiven immer wieder neu. Leitend ist dabei die These, dass die Landpartie im Romanwerk Fontanes einen Topos konstituiert, der stets dieselben Ordnungs- und Strukturprinzipien aufweist. Dieses Erzahlmuster wird in den einzelnen Texten allerdings sehr produktiv und variantenreich gestaltet. Kern der narrativen Gestaltung ist dabei stets eine Verknupfung von Liebes- und Geselligkeitshandlung. Dies verleiht der Landpartie handlungsdynamisierendes Potential, weshalb ihr eine wichtige konzeptionelle Funktion fur die Romane Fontanes zukommt.
Angesichts der Fülle von Beiträgen, die sich mit Theodor Fontanes Leben und Werk befassen, ist es erstaunlich, dass zur Landpartie bisher noch keine eigenständige Untersuchung vorliegt. Diese Studie ...widmet sich dem Thema erstmals umfassend und fokussiert es mit Blick auf sozial-, kultur- und medienhistorische Kontexte ebenso wie auf literatur- und kunsthistorische Perspektiven immer wieder neu. Leitend ist dabei die These, dass die Landpartie im Romanwerk Fontanes einen Topos konstituiert, der stets dieselben Ordnungs- und Strukturprinzipien aufweist. Dieses Erzählmuster wird in den einzelnen Texten allerdings sehr produktiv und variantenreich gestaltet. Kern der narrativen Gestaltung ist dabei stets eine Verknüpfung von Liebes- und Geselligkeitshandlung. Dies verleiht der Landpartie handlungsdynamisierendes Potential, weshalb ihr eine wichtige konzeptionelle Funktion für die Romane Fontanes zukommt.
Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis, and disease, bone morphogenetic proteins (BMPs) act as dimers to instruct ...specific cellular responses. BMP ligands can form both homodimers or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homodimers and heterodimers in the Drosophila wing imaginal disc. This approach identified in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb is secreted in a Dpp-dependent manner in the wing imaginal disc. Dpp and Gbb form a gradient of heterodimers, whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution.
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•Endogenous tagging combined with synthetic traps identifies BMP dimer composition in situ•Gbb secretion is dependent on Dpp expression•Gbb/Dpp heterodimers are the only BMP ligands secreted in the imaginal wing disc•Heterodimers trigger signaling at higher levels than would the Dpp homodimers
Bone morphogenetic proteins (BMPs) can potentially form both heterodimers or homodimers. Bauer et al. identify that in the Drosophila imaginal wing disc, heterodimers are the only secreted and fully active ligand species. In this tissue, the secretion of Gbb depends on its heterodimerization with Dpp.
CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of ...insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from “scarless editing by element deletion”), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
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•SEED/Harvest permits scarless tagging via single-strand annealing pathway in Drosophila•Tissue-specific Cas9 expression permits conditional endogenous tagging and mutant rescue•Tandem tagging strategy to simultaneously manipulate and visualize proteins•Engineered ALFA nanobody permits efficient live imaging of endogenously tagged proteins
Aguilar and Bauer et al. propose SEED (“scarless editing by element deletion”)/Harvest, a two-step gene-editing strategy that utilizes the SSA pathway to generate seamless knockins in Drosophila. SEED/Harvest allows for spatially restricted endogenous tagging and conditional rescues, mediated by tissue-specific Cas9 expression.
Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role ...of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.
The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the ...transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in
Drosophila melanogaster.
It binds to the
Drosophila
CSL homologue Suppressor of Hairless Su(H) and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the
Hairless
gene from
Daphnia pulex
and, hence, for the first time from a crustacean.
Daphnia
and
Drosophila
Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the
Daphnia
Hairless protein and the respective
Drosophila
interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between
Daphnia
Hairless and
Drosophila
Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during
Drosophila
development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from
Daphnia
and
Drosophila
are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.
