Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster.

M Alessandra Vigano,Manuela M M Kustermann,Gustavo Aguilar,Timothy J Stasevich,Markus Affolter,Shinya Matsuda,Ning Zhao,Clara-Maria Ell,George Pyrowolakis

doi:10.1242/dev.191700

M Alessandra Vigano, Manuela M M Kustermann + Show 7 more

Open Access

https://doi.org/10.1242/dev.191700

Copy DOI

Abstract

ABSTRACTCellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

Highlights

A key question in cell and developmental biology is how the millions of protein molecules present in any given cell regulate cellular functions in a predictable and coordinated manner
We wanted to investigate whether small tag binders [such as single chain fragments v and nanobodies (Nb)], which were shown to work in vivo as intrabodies, were able to bind single short peptide tags inserted in proteins located in different cellular compartments
We generated mammalian expression constructs for the anti-GCN4 (SunTag) single chain fragments v (scFv) (Tanenbaum et al, 2014), the anti-gp41(MoonTag) nanobody 2H10 (Boersma et al, 2019), the anti-HA scFvs (Zhao et al, 2019) and the anti-ALFA (Götzke et al, 2019) nanobody, each fused to either sfGFP or mEGFP for intracellular visualization

Summary

Introduction

A key question in cell and developmental biology is how the millions of protein molecules present in any given cell regulate cellular functions in a predictable and coordinated manner. Much of the work carried in the past decades to study protein function in their in vivo setting has relied on the use of genetic and reverse genetic approaches that, when combined with biochemical and structural. Handling Editor: Thomas Lecuit Received 18 April 2020; Accepted 9 February 2021 studies, have been extremely successful in gaining insight into protein function (Housden et al, 2017; Wang et al, 2016). It emerged that most proteins can interact with many different partners, often in a location- or context-dependent fashion, in many cases regulated by specific post-translational modifications. It would be desirable to have at hand a diversified toolbox to manipulate proteins directly in time and space in more controllable fashion

Methods

Results

Conclusion