Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.

Jonathan Cy Tang,Eugene Drokhlyansky,Sui Wang,Binggege Guo,Emily G Ellis,Jonathan Z Li,Stephanie Rudolph,Constance L Cepko,Behzad Etemad

doi:10.7554/elife.15312

Jonathan Cy Tang, Eugene Drokhlyansky + Show 7 more

Open Access

https://doi.org/10.7554/elife.15312

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Abstract

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.

Highlights

Many applications in biology and medicine require the ability to target a subset of cells in a population based upon specific cellular characteristics
To test whether it is possible to modify an Nb such that its intracellular protein level is strongly dependent upon antigen co-expression, we used the GFP-binding Nb, GBP1, for proof-of-concept experiments (Kirchhofer et al, 2010; Rothbauer et al, 2006) (Figure 1B,C)
We generated a Moloney murine leukemia virus (MMLV) library encoding randomly mutagenized variants of GBP1 fused to the blue fluorescent protein, TagBFP (Subach et al, 2008). t-HcRed (Gurskaya et al, 2001) was co-expressed via an IRES to report infection

Summary

Introduction

Many applications in biology and medicine require the ability to target a subset of cells in a population based upon specific cellular characteristics. This can be achieved by exploiting transcriptional elements that are selectively active in a subset of cells, specific elements are often not available, and can be difficult to generate. Other features that distinguish cells, such as expression of a specific RNA or protein, may be exploited. While current methods are promising, protein-responsive systems are continually evolving. There remains a need for generalizable strategies that enable rapid conversion of diverse classes of binders into protein-responsive tools

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