A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

Gerdes, Christoph; Waal, Natalia; Offner, Thomas; Fornasiero, Eugenio F.; Wender, Nora; Verbarg, Hannes; Manzini, Ivan; Trenkwalder, Claudia; Mollenhauer, Brit; Strohaeker, Timo; Zweckstetter, Markus; Becker, Stefan; Rizzoli, Silvio O.; Basmanav, Buket; Opazo, Felipe

doi:10.1101/846014

Cited by 8 publications

(9 citation statements)

References 97 publications

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“…Alternative methods to regulate the effective expression levels of the probe in tune with the one of its molecular targets would also be highly valuable for multiplexing applications as well as for systems (target, binder or cargo) outside of the optimal conditions mentioned above. Developing probes that undergo fast degradation unless bound to their target constitutes an interesting alternative that has been successfully used for the nanobody scaffold (Gerdes et al, 2020;Keller et al, 2018;Tang et al, 2016). Another strategy for imaging applications would consist in conditioning the resulting fluorescence rather than the probe stability to its target binding by the development of fluorogenic probes (Wongso et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Rimbault

Breillat

Compans

et al. 2021

Preprint

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Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

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Section: Discussionmentioning

confidence: 99%

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Rimbault

Breillat

Compans

et al. 2021

Preprint

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“…Monoclonal antibodies (mAbs) and antibody-derived biologics are essential tools for cancer research and Strohl, 2018;Nikooharf et al, 2020;Kijanka et al, 2015 Direct antagonistic effects (bind to extracellular proteins) Farrants et al, 2020;Kirchhofer et al, 2010 Nbs incorporated into drug delivery systems Muhammad et al, 2017;Oliveira et al, 2010 Study protein-protein interactions in vivo De Meyer et al, 2014;Herce et al, 2013 Nbs used for in vivo medical imaging Rothbauer et al, 2006;Roovers et al, 2007;Platonova et al, 2015;Wang et al, 2016;Prole and Taylor, 2019;Salvador et al, 2019;Sograte-Idrissi et al, 2019 For tracing intracellular targets in various compartments in living cells Rothbauer et al, 2006;Beghein and Gettemans, 2017;Traenkle and Rothbauer, 2017;Debie et al, 2019 Nbs used as diagnostics Pereira et al, 2014;Hoey et al, 2019 Nanobodies against inflammation and autoimmune diseases Tanaka et al, 2014;Sadeghian-Rizi et al, 2019 Nbs against hematological disorders Peyvandi et al, 2016;Scully et al, 2019 Nbs against viruses Vanlandschoot et al, 2011;Cardoso et al, 2014;Ezzikouri et al, 2020;Hanke et al, 2020. Nbs for allergy treatment Flicker et al, 2020 Nbs against neurodegenerative diseases Messer and Butler., 2020;Gerdes et al, 2020 Nbs: nanobodies (Griffiths et al, 2018) Nanoantibodies: a narrative review 329 therapy (Strohl, 2018). Antibodies can be used to inhibit tumor cell proliferation and as tools to define effector domains.…”

Section: Nbs In Cancer Therapymentioning

confidence: 99%

Nanoantibodies: small molecules, big possibilities

Pedreáñez¹,

Mosquera-Sulbarán²,

Muñoz³

et al. 2021

bta

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Camelids (camels, dromedaries, alpacas, llamas, and vicuZas) contain in their serum conventional heterodimeric antibodies as well as antibodies with no light chains (L) in their structure and composed of only heavy chains (H), called as HcAbs (heavy chain antibodies). Variable fragments derived from these antibodies, called as VHH or nanoantibodies (Nbs), have also been described. Since their discovery, Nbs have been widely used in the fields of research, diagnostics, and pharmacotherapy. Despite being approximately one-tenth the size of a conventional antibody, they retain similar specificity and affinity to conventional antibodies and are much easier to clone and manipulate. Their unique properties such as small size, high stability, strong antigen binding affinity, water solubility, and natural origin make them suitable for the development of biopharmaceuticals and nanoreagents. The present review aims to describe the main structural and biochemical characteristics of these antibodies and to provide an update on their applications in research, biotechnology, and medicine. For this purpose, an exhaustive search of the biomedical literature was performed in the following databases: Medline (PubMed), Google Scholar, and ScienceDirect. Meta-analyses, observational studies, review articles, and clinical guidelines were reviewed. Only original articles were considered to assess the quality of the evidence.

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“…Nbs have unique properties relative to conventional antibodies that make them attractive for use as research tools and development of therapeutics (Steeland et al, 2016). Nbs are small (15 kDa), have high chemical stability, can be genetically-encoded for expression in mammalian cells as intrabodies, and they can bind hidden epitopes in small cavities that are not recognized by antibodies (Li et al, 2016; Beghein and Gettemans, 2017; Dong et al, 2019; Gerdes et al, 2020; Muyldermans, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Nbs are small (15 kDa), have high chemical stability, can be genetically-encoded for expression in mammalian cells as intrabodies, and they can bind hidden epitopes in small cavities that are not recognized by antibodies (Li et al, 2016;Beghein and Gettemans, 2017;Dong et al, 2019;Gerdes et al, 2020;Muyldermans, 2021).…”

Section: Introductionmentioning

confidence: 99%

Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

Ishizuka

Mergiya

Baldinotti

et al. 2022

Preprint

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Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

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A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

Cited by 8 publications

References 97 publications

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Nanoantibodies: small molecules, big possibilities

Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

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