A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons

Dong, Jie; Lee, Yongam; Kirmiz, Michael; Palacio, Stephanie; Dumitras, Camelia; Moreno, Cláudia; Sando, Richard; Santana, Luis Fernando; Südhof, Thomas C.; Gong, Belvin; Murray, Karl D.; Trimmer, James S.

doi:10.7554/elife.48750

Cited by 46 publications

(60 citation statements)

References 70 publications

(93 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Exorbitant expression levels can be circumvented by a replacement strategy in which a tagged protein is expressed in a knock-down or knock-out background [9], but this will, at best, only approximate endogenous levels and is uncoupled from endogenous transcriptional or translational regulatory mechanisms. Recombinant antibody-based approaches have been developed for live-cell imaging of neuronal proteins, but they have so far been restricted to a few targets [10][11][12][13][14]. The generation of fluorescently tagged knock-ins (for instance, in mouse lines) prevents these issues.…”

Section: Introductionmentioning

confidence: 99%

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

et al. 2020

View full text Add to dashboard Cite

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knockins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

show abstract

Section: Introductionmentioning

confidence: 99%

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

et al. 2020

View full text Add to dashboard Cite

show abstract

“…The applications are multiple. For instance, fluorescent nanobodies can be expressed at a determined physiological phase of the host cells to follow the fate of the corresponding antigens without interfering with their activity and by such a way specifically labeling distinct antigens and subcellular regions [48]. The clear advantage of nanobodies over conventional antibodies is their structural simplicity with a single disulfide bond that in several cases is not obligatory for reaching stable Both heavy-and light-chain variable domains contribute to the antigen-selective binding surface of conventional IgGs, whereas the heavy-chain variable domain of Camelidae IgGs provides alone the complete epitope for the antigen specific recognition.…”

Section: How To Choose Between Mammalian and Bacterial Expression Sysmentioning

confidence: 99%

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

View full text Add to dashboard Cite

A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

show abstract

“…Gene therapy via the AAV-mediated delivery of an anti-Aβ42 intrabody in an AD mouse model allowed partial clearance of Aβ42 deposits [155], and in a follow-up study, myelin integrity was restored in mice treated with intrabody gene therapy [156]. An intrabody was furthermore used for imaging purposes in live neurons to study mechanisms of memory impairment in AD [157], and intrabodies have been found to be generally valuable tools for imaging purposes in neurons [158]. AD is also associated with abnormal tau phosphorylation as well as aggregation and is the most common tauopathy [159,160].…”

Section: Alzheimer's Diseasementioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

View full text Add to dashboard Cite

To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

show abstract

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons

Cited by 46 publications

References 70 publications

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

Contact Info

Product

Resources

About