Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies ...have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms.
p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The ...ΔNp63α isoform is thought to play a ‘master’ role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing ΔNp63α; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.
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.
Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies ...to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms.
Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in ...cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using
, we also find that single short tags can be used for POI manipulation
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Summary Background An established multivariate serum protein test can be used to classify patients according to whether they are likely to have a good or poor outcome after treatment with EGFR ...tyrosine-kinase inhibitors. We assessed the predictive power of this test in the comparison of erlotinib and chemotherapy in patients with non-small-cell lung cancer. Methods From Feb 26, 2008, to April 11, 2012, patients (aged ≥18 years) with histologically or cytologically confirmed, second-line, stage IIIB or IV non-small-cell lung cancer were enrolled in 14 centres in Italy. Patients were stratified according to a minimisation algorithm by Eastern Cooperative Oncology Group performance status, smoking history, centre, and masked pretreatment serum protein test classification, and randomly assigned centrally in a 1:1 ratio to receive erlotinib (150 mg/day, orally) or chemotherapy (pemetrexed 500 mg/m2 , intravenously, every 21 days, or docetaxel 75 mg/m2 , intravenously, every 21 days). The proteomic test classification was masked for patients and investigators who gave treatments, and treatment allocation was masked for investigators who generated the proteomic classification. The primary endpoint was overall survival and the primary hypothesis was the existence of a significant interaction between the serum protein test classification and treatment. Analyses were done on the per-protocol population. This trial is registered with ClinicalTrials.gov , number NCT00989690. Findings 142 patients were randomly assigned to chemotherapy and 143 to erlotinib, and 129 (91%) and 134 (94%), respectively, were included in the per-protocol analysis. 88 (68%) patients in the chemotherapy group and 96 (72%) in the erlotinib group had a proteomic test classification of good. Median overall survival was 9·0 months (95% CI 6·8–10·9) in the chemotherapy group and 7·7 months (5·9–10·4) in the erlotinib group. We noted a significant interaction between treatment and proteomic classification (pinteraction =0·017 when adjusted for stratification factors; pinteraction =0·031 when unadjusted for stratification factors). Patients with a proteomic test classification of poor had worse survival on erlotinib than on chemotherapy (hazard ratio 1·72 95% CI 1·08–2·74, p=0·022). There was no significant difference in overall survival between treatments for patients with a proteomic test classification of good (adjusted HR 1·06 0·77–1·46, p=0·714). In the group of patients who received chemotherapy, the most common grade 3 or 4 toxic effect was neutropenia (19 15% vs one <1% in the erlotinib group), whereas skin toxicity (one <1% vs 22 16%) was the most frequent in the erlotinib group. Interpretation Our findings indicate that serum protein test status is predictive of differential benefit in overall survival for erlotinib versus chemotherapy in the second-line setting. Patients classified as likely to have a poor outcome have better outcomes on chemotherapy than on erlotinib. Funding Italian Ministry of Health, Italian Association of Cancer Research, and Biodesix.
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.
p63 is a transcription factor with a "master" role in the asymmetric cell division of stratified epithelia. The transcriptional strategy is exerted by activating and repressing a wide range of genes. ...Our understanding of the pathways and networks controlled by p63 is starting to emerge, thanks to profiling arrays and ChIP on chip experiments. We discuss recent advancements in the identification of bona fide targets, which suggests that several independent, as well as interconnected pathways are controlled by p63. Not surprisingly, the list includes genes previously shown to play a key role in differentiation processes, as well as targets involved in cell-cycle control, signalling and transcription.
Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With ...regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology.
This article is categorized under:
Technologies > Analysis of Proteins
Establishment of Spatial and Temporal Patterns > Gradients
Invertebrate Organogenesis > Flies
Protein Quest: Guidelines for the use of protein binders in developmental biology.
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