Hexanediol can differentiate between liquid- and solid like assemblies in vitro
Previous studies have used the aliphatic alcohol 1,6-hexanediol to test the material properties of cellular assemblies. Hexanediol was shown to perturb FG repeat interactions between nucleoporins in nuclear pores and interactions between RNA-binding proteins in RNA-protein granules (RNP). These studies suggested that 1,6-hexanediol interferes with weak hydrophobic protein-protein or protein-RNA interactions that are required for these dynamic, liquid-like assemblies to form. A well characterized protein that forms liquid-like compartments is FUS. FUS is primarily nuclear and involved in transcription, DNA repair and RNA processing. The protein also relocates to stress granules upon environmental perturbations. In both conditions, FUS forms compartments that have liquid-like properties. It was further reported that liquid-like FUS compartments undergo a liquid-to-solid transition in patients afflicted with ALS and related diseases. This makes FUS an ideal candidate protein to test how 1,6-hexanediol affects two different material states formed by the same protein. To test the utility of hexanediol, we reconstituted FUS compartments as reported previously using dextran to mimic cellular crowding conditions (Panel A, top left). The formed droplets dissolved quickly upon the addition of 4% hexanediol, in contrast to the buffer-only control (Panel A, bottom). Next, we tested if hexanediol also affects the solid-like state of FUS. Solid FUS fibers form in an in vitro aging assay after 24 h (Panel A, top left). The fibers were incubated with 4% hexanediol (Panel A, bottom right). However, no dissolution was observed (Panel A). We therefore conclude that hexanediol can be used to differentiate liquid-like and solid-like states of protein assemblies.
Liquid-like but not solid-like assemblies in living yeast cells are vulnerable to hexanediol
To evaluate whether hexanediol can differentiate between liquid- and solid-like assemblies in living yeast cells, we first determined the ideal concentration range for the use of hexanediol. We found that hexanediol concentrations of 10% quickly dissolved P bodies in budding yeast cells, whereas 5% hexanediol only dissolved P bodies after 20–30 min. These experiments were performed in the presence of 10 µg/ml digitonin, which makes yeast cells more permeable to hexanediol (Panel B). Thus, we recommend using hexanediol at a concentration of 10% together with digitonin. Please note that in some cells assemblies reappear after 30–60 min. This will be addressed in the next section.
To determine whether hexanediol can be used to probe the material properties of cellular structures, we next tested several assemblies with more solid-like character for their sensitivity to hexanediol. We first looked at protein aggregates. Protein aggregates were induced through glucose starvation, and the chaperone Hsp104 was used as a marker. Prominent foci of Hsp104-positive aggregates were visible after 1 h. These Hsp104-positive protein aggregates were resistant to hexanediol (Panel C). This is in agreement with the finding that yeast stress granules, which colocalize with Hsp104 and depend on Hsp104 for dissolution, are insensitive to hexanediol (Panel C). We next tested whether actin filaments and microtubules are affected by hexanediol treatment (Panel D). However, the hexanediol effects on the integrity of these cytoskeletal structures were mild and did not lead to a dissolution of actin filaments or microtubules. Thus, more solid-like protein assemblies such as protein aggregates and the cytoskeleton are not affected by hexanediol when used at a concentration of 10% in the presence of digitonin.
A previous study reported a partial dissolution of stress granules and cytoskeletal structures upon hexanediol treatment. However, under the conditions that we used, we did not find evidence for a hexanediol sensitivity of these structures. This may partially be due to the use of different growth and hexanediol treatment conditions. Another factor that can affect the outcome is the duration of the stress stimulus prior to the hexanediol treatment. This sensitivity to conditions (hexanediol concentration, stress period, growth conditions) should be taken into account when analyzing membrane-less compartments and assemblies with hexanediol.
We conclude that hexanediol can differentiate between liquid-like and more solid-like assemblies in yeast cells. However, the specific conditions under which the hexanediol experiment is performed are very important and will strongly impact the obtained results. We therefore recommend performing comparative experiments with assemblies for which the material properties are known.
Extended exposure of cells to hexanediol leads to loss of membrane integrity
When we performed extended time-lapse experiments in the presence of hexanediol, we have noticed that some yeast cells, which had initially lost P bodies, re-accumulated P body-like structures at a later time point (Panel E). This has been also observed by others. However, these structures also colocalized with SG markers (Panel H), something that is normally only observed in cells that are exposed to severe stress. To investigate the viability of these cells, we used the dye trypan blue. This dye accumulates within cells that have lost their membrane integrity and can thus be used to differentiate between live and dead cells. Interestingly, the induction of late P body-like structures coincided with the influx of trypan blue into the cells (Panel E, bottom and Panel F). Loss of membrane integrity, as indicated by the accumulation of trypan blue within cells, also coincided with cell shrinkage (Panel G). This indicates that, upon prolonged exposure to hexanediol, yeast cells die. This results in the formation of aberrant structures that contain both P body and stress granule components.
We hypothesized that these assemblies form because the permeabilized cells experience altered osmotic conditions that lead to significant cell shrinkage and increased macromolecular crowding. To investigate this, we exposed yeast to a hypertonic shock. Indeed, under hypertonic conditions, we observed similar assemblies, which were positive for PB proteins (Panel H). Note that this hypertonic shock was applied in the absence of hexanediol. Importantly, hypotonic media delayed the formation of these aberrant assemblies in the presence of hexanediol (Panel H, compare top and bottom). Thus, we conclude that extended hexanediol treatment can lead to loss of membrane integrity and osmotic compression that causes the formation of aberrant assemblies in yeast cells. For this reason, we recommend performing live cell time-lapse imaging to separate early effects of hexanediol from later aberrant effects due to osmotically induced cell shrinkage. Time-resolved imaging will allow monitoring of individual cells and will reveal sudden changes in cellular morphology, and the appearance of aberrant aggregates with time. We also propose including vital dyes in the experiment such as trypan blue. These dyes can be added together with hexanediol and this will aid in identifying cells with compromised membrane integrity.
Similar to yeast cells, liquid-like assemblies in mammalian cells are dissolved through hexanediol
Similar to yeast P bodies, mammalian stress granules dissolve in a hexanediol-dependent manner over a time course of 10 to 20 min. This is due to the fact that stress granules in mammalian cells are much more liquid-like than stress granules in yeast cells. To further investigate this, we tested the concentration dependence of the hexanediol effect. The dissolution of mammalian stress granules could be observed with hexanediol concentrations of 3.5% and higher (Panel I). Some cells also re-accumulated smaller stress granule-like structures labeled by G3BP2 under hexanediol treatment, which were especially prominent in unstressed cells, but these structures dissolved again (Panel J). This is reminiscent of the situation that we observed upon prolonged exposure of yeast cells to hexanediol, where the cells lost membrane integrity with time and formed aberrant structures.
To test whether the appearance of these late structures is due to increased membrane permeability and cell shrinkage and whether osmotic effects are involved, we repeated the experiment in hypotonic media. The cells were first imaged in regular media (time point “before”) and subsequently the media was replaced with hypotonic media containing hexanediol (Panel K, top). Indeed, in hypotonic media, aberrant stress granule-like structures did not form in the presence of hexanediol, while in hypertonic media stress granule-like structures formed even without hexanediol addition (Panel K, bottom). This suggests that, similar to the situation in yeast cells, the formation of aberrant assemblies under extended hexanediol treatment is caused by a loss of membrane function and a potential loss of water from the cells that leads to an increase in macromolecular crowding. Thus, we conclude that hexanediol can be used to determine the material state of cellular assemblies in mammalian cells, but the extended exposure to hexanediol should be avoided.
1,6-hexanediol induces blebbing in HeLa cells, but it does not cause cell death
In contrast to yeast cells, HeLa cells are much more vulnerable to environmental changes. Exposure to adverse conditions often leads to the formation of membrane blebs, which can be the first signs of autophagy. Indeed, we noticed that the addition of hexanediol to HeLa cells caused blebbing. However, cells that had been incubated with hexandiol for as long as 50 min could recover from blebbing and spread again when hexanediol was washed out and replaced with normal growth media (Panel L). Thus, incubation with hexanediol does not cause the immediate death of cultured mammalian cells such as HeLa cells, when the cells are incubated with hexanediol for a limited amount of time.