commentary

Five challenges to bringing single-molecule force into living cells Yves F Dufrêne, Evan Evans, Andreas Engel, Jonne Helenius, Hermann E Gaub & Daniel J Müller

In recent years, single-molecule force spectroscopy techniques have been used to study how inter- and intramolecular interactions control the assembly and functional state of biomolecular machinery in vitro. Here we discuss the problems and challenges that need to be addressed to bring these technologies into living cells and to learn how cellular machinery is controlled in vivo.

The living is a highly specialized factory problems and challenges that need to be processes and what feedback these pro- that has evolved ways to precisely control addressed to quantify interactions of single cesses induce requires characterizing these inter- and intramolecular interactions that molecules in vivo. mechanisms in the living cell (Fig. 2). regulate its molecular machinery. But how Accordingly, we have to develop methods cells establish such biomolecular interac- Cellular complexity defines analytical that allow single to be force- tions remains mysterious. Single-molecule approaches probed in vivo. This will entail bringing force spectroscopy (SMFS) devices have Biomolecular interactions can generate tiny together nanoscale science, , opened up a wide range of opportunities to forces in the piconewton range that persist engineering, and molecular and cell biolo- quantify and manipulate the interactions of over time spans from milliseconds to many gy. Here we attempt to define five big needs individual biomolecules. Nowadays, SMFS minutes or more. In the last two decades, that will allow SMFS to reach this goal. is increasingly used to explore the folding, tremendous progress has been made in Some of these challenges overlap with those conformational entropy, mobility, assembly developing SMFS methods (Fig. 1a) that for applying SMFS in vitro. Therefore, we and functionalities of molecular machinery can be used to quantify such forces that pay particular attention to the differences of the cell in vitro. These SMFS studies have contribute to cellular and molecular sys- in addressing the challenges of in vitro to an inherent flaw: they use purified biomol- tems1–6. SMFS has provided insights into in vivo systems.

Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature © 201 ecules that are removed from the cellular how biomolecular interactions guide pro- context. However, deciphering how cells tein folding, stability and functional state, Define and overcome the limitations of control biomolecular interactions to drive macromolecular assembly, ligand and current tools their machinery in the highly complex inhibitor binding, molecular transport, There is a clear need to define the limita- and dynamic environment of their interior signal transduction, mechanosensing and tions of our research tools for force-probing requires transferring SMFS into a living cell. cell adhesion, motility, sorting and differen- single molecules in the cellular context. Only with such a paradigm change will the tiation. But nearly all current SMFS assays Historically, SMFS has been developed combination of in vitro and in vivo SMFS probe interactions of isolated biomolecules mainly to manipulate and probe isolated measurements provide key insights into in vitro (Fig. 1b). Because biomolecules are biomolecular systems such as receptor- cellular processes. Here we scrutinize the highly controlled by their cellular environ- ligand pairs, DNA, membranes and pro- ment, their interactions with the environ- teins4,10–12. The SMFS methods used were Yves F. Dufrêne is at Universite catholique de Louvain, ment are different in the living cell than based either on atomic force In