1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells.

Yuji Itoh,Sachiko Tamura,Kayo Hibino,Tatsuhiko Goto,Ryosuke Nagashima,Shiori Iida,Satoru Ide,Kentaro Shiraki,Kazuhiro Maeshima

doi:10.26508/lsa.202001005

Abstract

Liquid droplets formed inside the cell by liquid-liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein-protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was "frozen" by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.

Highlights

Some macromolecules self-organize into liquid droplets by a process termed liquid–liquid phase separation (LLPS), which allows specific molecules to be concentrated without a membrane, whereas others are excluded (Hyman et al, 2014; Banani et al, 2017; Shin & Brangwynne, 2017)
Our single-nucleosome imaging/tracking revealed that the aliphatic alcohol 1,6-HD, which has been widely used for LLPS studies, can immobilize chromatin motion and hyper-condense chromatin in live cells
Another aliphatic alcohol, 2,5-HD, which has a much lower melting activity of droplets formed by LLPS (Lin et al, 2016), had a comparable motion suppression effect to 1,6-HD (Fig 3D)

Summary

Introduction

Some macromolecules self-organize into liquid droplets by a process termed liquid–liquid phase separation (LLPS), which allows specific molecules to be concentrated without a membrane, whereas others are excluded (Hyman et al, 2014; Banani et al, 2017; Shin & Brangwynne, 2017). Cells organize liquid droplet-like condensates/bodies (or compartments), contributing to the spatial and temporal regulation of complex biochemical reactions. Whether all of these dynamic biomolecular condensates/bodies form by LLPS or some form by another process remains unclear and the subject of debate for many cell biologists (McSwiggen et al, 2019). The aliphatic alcohol, 1,6-hexanediol (1,6-HD) (Fig 1A), has been widely used to study the formation process of the membrane-less cytoplasmic/ nuclear condensates/bodies, presumably formed by LLPS (Kroschwald et al, 2017). We confirmed several published results that 1,6-HD treatment disrupted nuclear condensates/bodies (Cho et al, 2018; Lin et al, 2016), we found that 1,6-HD drastically and globally suppressed chromatin motion and hyper-condensed chromatin in live HeLa cells. Careful consideration is needed to interpret all the results of cell biological experiments performed with 1,6-HD treatment

Results

Discussion

Findings

Materials and Methods