Nanobody System Tags, Grabs, Drags Proteins in the Cell

A nanobody-based system has been used to control intracellular protein localization and enable detailed investigation of wing development in the fruit fly. The system, its developers speculate, may help illuminate diverse mechanisms in developmental biology. In this image, nanobodies in the wing precursor of a fruit fly larva are shown in pink. [University of Basel, Biozentrum]

An object’s function depends not only on its size, shape, and composition, but its location, too—as anyone who has ever been asked to arrange furniture knows. Moving a couch here or a table there can even alter the function of an entire room. Something similar can be said about proteins, and their positions within the cell. Yet the relocalization or even the deliberately mislocalization of proteins isn’t easily accomplished.

To accomplish the cellular equivalent of furniture arrangement, scientists based at the University of Basel have developed a nanobody-based system that can accomplish protein localization. The system, its creators assert, can be used with a wide range of proteins and in various areas of developmental biology.

The Basel team, led by developmental biologist Markus Affolter, Ph.D., used the nanobody system to investigate the growth of the wings of the fruit fly. Details of this work appeared April 11 in the journal eLife, in an article entitled “A Nanobody-Based Toolset to Investigate the Role of Protein Localization and Dispersal in Drosophila.”

According to the article’s authors, the nanobody system, called GrabFP (for grab Green Fluorescent Protein), is a collection of four nanobody-based green fluorescent protein (GFP) traps that localize to defined positions along the apical–basal axis. The localization preference of the GrabFP traps, they explained, can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization.

“These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization,” they wrote. “Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.”

A repositioning of the proteins of interest requires labeling with GFP. Subsequently, so-called anti-GFP nanobodies, small antibody fragments derived from camels, are then used to bind and to move the GFP-tagged proteins to a new site in the living organism. The nanobody itself is linked to a signal protein that defines the destination of the target protein. Thus, the nanobody forces the GFP-tagged protein into a new position. “Even if we do not know exactly the composition and structure of a protein, we can label it with GFP and control the destination site by using nanobodies,” said Stefan Harmansa, Ph.D., one of the two first authors.

The researchers were able to transfer proteins to a new site, internal or external to the cell. “By transporting proteins to new locations, we can observe whether their function changes or not and whether development is affected,” says Ilaria Alborelli, Ph.D., also one of the first authors of the study.

So far, scientists have been restricted in relocating proteins. The new nanobody tool, however, makes it possible to easily and efficiently change the position of all GFP-tagged proteins and thus explore their functions. The Affolter group has already been successful in investigating the growth of Drosophila wings using this nanobody tool. By interfering with the signaling molecule Dpp in a position-dependent manner, the scientists have been able to show more precisely its influence on wing growth.

In the future, the new nanobody tool can be used for a wide variety of studies on organ growth and in various other areas of developmental biology, and the growth and the development of different cells and organs can be investigated in more detail.

The Affolter team also faces many new challenges, noted Harmansa: “We as developmental biologists are still confronted with urgent questions, such as how an organism knows when it has to stop its growth. To put it succinctly, how does it work that arms or legs stop growing when they reach their correct length?” In the future, this novel tool may contribute to a better understanding of how organ growth is regulated.