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  • 1
    In: Development, The Company of Biologists, Vol. 148, No. 6 ( 2021-03-15)
    Abstract: 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 Drosophila, we also find that single short tags can be used for POI manipulation in vivo.
    Type of Medium: Online Resource
    ISSN: 0950-1991 , 1477-9129
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2021
    detail.hit.zdb_id: 2007916-3
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    MDPI AG ; 2019
    In:  Antibodies Vol. 8, No. 1 ( 2019-02-12), p. 16-
    In: Antibodies, MDPI AG, Vol. 8, No. 1 ( 2019-02-12), p. 16-
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 2073-4468
    Language: English
    Publisher: MDPI AG
    Publication Date: 2019
    detail.hit.zdb_id: 2661514-9
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  • 3
    Online Resource
    Online Resource
    Wiley ; 2019
    In:  WIREs Developmental Biology Vol. 8, No. 6 ( 2019-11)
    In: WIREs Developmental Biology, Wiley, Vol. 8, No. 6 ( 2019-11)
    Abstract: 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
    Type of Medium: Online Resource
    ISSN: 1759-7684 , 1759-7692
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2649746-3
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  • 4
    In: Biology Open, The Company of Biologists, Vol. 7, No. 12 ( 2018-12-15)
    Type of Medium: Online Resource
    ISSN: 2046-6390
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2018
    detail.hit.zdb_id: 2632264-X
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  • 5
    In: Biology Open, The Company of Biologists
    Abstract: Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.
    Type of Medium: Online Resource
    ISSN: 2046-6390
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2018
    detail.hit.zdb_id: 2632264-X
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