A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

Jessica Colombo,Pekka Lappalainen,Alphée Michelot,Tommi Kotila,Adrien Antkowiak,Audrey Guillotin,Jenna Elliott,Konstantin Kogan

doi:10.1038/s41467-020-20827-4

Abstract

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

Highlights

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of highquality probes
This study identifies a family of fluorescent nucleotides, N6-(6Amino)hexyl-adenosine triphosphate (ATP) derivatives, which binds to actin
In-depth characterization of this interaction demonstrates that these probes do not interfere with actin polymerization, and do not prevent the interactions of actinbinding proteins (ABPs) such as profilin, actin-depolymerizing factor (ADF)/cofilin or tropomyosin with actin

Summary

Introduction

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of highquality probes. We identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. The Arp2/3 complex accelerates nucleation of new actin filaments from the sides of pre-existing mother filaments, whereas heterodimeric capping protein controls the elongation of actin filaments at their rapidly growing barbed ends[6] Other proteins such as actin-depolymerizing factor (ADF)/ cofilin sever actin filaments to enhance their disassembly[9,10], whereas profilin and cyclase-associated protein accelerate the nucleotide exchange on the resulting ADP actin monomers, thereby contributing in replenishing the cellular pool of ATP-Gactin[11,12,13]. There is a need for robust approaches for visualizing actin filaments

Methods

Results

Conclusion