Mapping cytoskeletal protein function in cells by means of nanobodies

Abstract

Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection.

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

“…Depending on their binding properties they can be used to visualize subcellular antigen location or to modulate their target structure and function (31,33,43,55,71). Here, we have demonstrated that all selected nanobodies are able to bind ␤-catenin in living cells.…”

“…Intracellular Application of ␤-catenin Chromobodies-Recently, we and others have shown that V H H domains derived from heavy chain antibodies of camelids can be selected as intrabodies for functional interference and visualization of target structures (31,33,54,55). To detect endogenous ␤-catenin within living cells, we fused the coding sequences of the nanobodies to the fluorescent protein tagGFP generating so-called chromobodies (BC-chromobodies) and introduced them on DNA-level either by transfection or viral transduction in living cells.…”

Biophysical characterization of antibodies with isothermal titration calorimetry

“…Nanobodies are emerging as versatile tools in biotechnology. Several recent studies incorporated ITC to characterize nanobodies function and structure [55][56][57].…”

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

“…Moreover, in this way, one can also disturb protein function by restricting free diffusion of the protein and limiting its availability at places where it is needed [71]. Considering that the paratope of the nanobody is located at its N-terminal end, it is safer to fuse the tag at the C-terminal end of the nanobody.…”