Abstract
ABSTRACTLife is dependent upon the ability of a cell to rapidly respond to changes in the environment. Small perturbations in local environments change the ability of molecules to interact and, hence, communicate. Hydrostatic pressure provides a rapid non-invasive, fully reversible method for modulating affinities between molecules both in vivo and in vitro. We have developed a simple fluorescence imaging chamber that allows intracellular protein dynamics and molecular events to be followed at pressures <200 bar in living cells. By using yeast, we investigated the impact of hydrostatic pressure upon cell growth and cell-cycle progression. While 100 bar has no effect upon viability, it induces a delay in chromosome segregation, resulting in the accumulation of long undivided cells that are also bent, consistent with disruption of the cytoskeletons. This delay is independent of stress signalling and induces synchronisation of cell-cycle progression. Equivalent effects were observed in Candida albicans, with pressure inducing a reversible cell-cycle delay and hyphal growth. We present a simple novel non-invasive fluorescence microscopy-based approach to transiently impact molecular dynamics in order to visualise, dissect and study signalling pathways and cellular processes in living cells.
Highlights
All life is dependent upon the ability of a cell to rapidly respond to changes in its environment through modulation of diverse signalling pathways
In mid-log phase at 25°C, were placed in the pressure chamber and exposed to elevated pressure for times between 1 and 24 h before pressure was returned to 1 bar, and samples were collected for viewing using standard microscopy or were plated out to assess viability
In a final investigation into the effect of hydrostatic pressure on yeast growth dynamics, we investigated the impact pressure has upon the growth of a different yeast cell, the pathogenic budding yeast Candida albicans
Summary
All life is dependent upon the ability of a cell to rapidly respond to changes in its environment through modulation of diverse signalling pathways. Small perturbations in local environments change the ability of molecules to interact and, communicate. Hydrostatic pressure provides a rapid non-invasive and fully reversible method to modulate the affinities between molecules both in vivo and in vitro. Hydrostatic pressure is a powerful tool to perturb protein–protein and protein–ligand interactions in complex environments. It has been widely used to study proteins and membranes in solution Barriga et al, 2016; Brooks et al, 2011; Coates et al, 1985; Eccleston et al, 1988) but less so in cellular systems.
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