Using Nanobodies to Study Protein Function in Developing Organisms

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.

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

“…Another functionalization that can be applied to the small tag binders is the addition of “degrons” (Natsume and Kanemaki, 2017) to achieve specific and temporally-controlled degradation of the tagged POIs. This approach has been successfully applied using the anti-FP nanobodies (Aguilar et al, 2019a; Beghein and Gettemans, 2017; Deng et al, 2020; Ingram et al, 2018; Prole and Taylor, 2019). Here we expand these findings by demonstrating that a single short HA tag can be efficient applied to inactivate POIs using deGradHA, a tool adapted from the deGradFP system and designed to channel HA-tagged POIs to ubiquitin/proteasome dependent degradation.…”

“…These protein binders can be functionalized to allow the regulation of POIs in a desired manner. Using functionalized protein binders, POIs can be visualized, degraded, delocalized, or post-transcriptionally modified in vivo in order to learn more about the function of the POIs in cultured cells or in developing organisms (Aguilar et al, 2019a; Bieli et al, 2016; Harmansa and Affolter, 2018; Helma et al, 2015; Holliger and Hudson, 2005; Pluckthun, 2015; Prole and Taylor, 2019; Schumacher et al, 2018).…”

“…Ideally, and to minimize the potential perturbation of the POI, the latter could be tagged by a short peptide to which high affinity protein binders have been identified and characterized; this approach would allow the use of characterized and validated binders and results in minimal potential disturbance of the function of the POI. Multiple protein manipulation tools generated with nanobodies or DARPins directed towards FPs (Aguilar et al, 2019a; Beghein and Gettemans, 2017; Brauchle et al, 2014; Schumacher et al, 2018; Vigano et al, 2018) could be adapted in order to functionalize small tag binders.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The possibility to use intrabodies to selectively modify the activity of a target protein for studying its function in vivo in eukaryotic cells has been achieved by tagging the target proteins and using them in combination with nanobodies that recognize the tag [52]. Anti-tag nanobodies with intrabody features have been successfully exploited to induce visualization, degradation, relocalization, trapping and modification of the protein of interest [53]. The deGradFP system exploits an anti-GFP-Fbox fusion nanobody that promotes the ubiquitination of the bound antigen and its consequent degradation mediated by the proteasome machinery.…”

Special Issue: Nanobody

“…This set of original work is further complemented by a series of reviews highlighting the emerging potential of nanobodies in biomedical research, diagnostics and therapy. Aguilar and colleagues, the pioneers in the field, summarized recent developments on how intracellularly functional nanobodies combined with functional or structural units can be used to study and manipulate protein function in multicellular organisms and developmental biology [17]. As exemplified by several studies in this Special Issue, nanobodies open new avenues for the treatment of viral infections.…”