Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging.

While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and ß-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated ß-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation.

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (~ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Nanobodies targeting cortactin proline rich, helical and actin binding regions downregulate invadopodium formation and matrix degradation in SCC-61 cancer cells.

Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level. Two cortactin nanobodies target the central region of cortactin with high specificity. One nanobody interacts with the actin binding repeats whereas the other targets the proline rich region and was found to reduce EGF-induced cortactin phosphorylation. After intracellular expression as an intrabody, they are both capable of tracing their target in the complex environment of the cytoplasm, and disturb cortactin functions during invadopodia formation and extracellular matrix degradation. These data illustrate the use of nanobodies as a research tool to dissect the role of cortactin in cancer cell motility. This information can contribute to the development of novel therapeutics for tumor cell migration and metastasis.

Reviving old protecting group chemistry for site-selective peptide-protein conjugation.

Methodologies to conjugate proteins to property-enhancing entities are highly sought after. We report a remarkably simple strategy for conjugating proteins bearing accessible cysteines to unprotected peptides containing a Cys(Scm) protecting group, which is introduced on-resin via a Cys(Acm) building block. The peptides employed for this proof of principle study are highly varied and structurally diverse, and undergo multiple on-resin decoration steps prior to conjugation. The methodology was applied to three different proteins, and proved to be efficient and site-selective. This twist on protecting group chemistry has led to a novel and generally applicable strategy for crossed-disulfide formation between proteins and peptides.

VCA nanobodies target N-WASp to reduce invadopodium formation and functioning.

Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp-Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes.