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Macromolecular condensation buffers intracellular water potential

ABSTRACT: Optimum protein function and biochemical activity critically depends on water availability, because solvent thermodynamics drive protein folding and macromolecular interactions. Reciprocally, macromolecules restrict the movement of “structured” water molecules within their hydration layers, reducing the available “free” bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Within concentrated macromolecular solutions like the cytosol, we found modest changes in temperature greatly impact the water potential, and are counteracted by opposing changes in osmotic strength. Remarkably, this duality of temperature and osmotic strength allows simple manipulations of solvent thermodynamics to prevent cell death upon extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength that impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales, so we postulated the existence of a rapid compensatory response. We find this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically-disordered proteins. Formation or dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which in consequence is robustly buffered in the cytoplasm. Our results indicate biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Lung, Fibroblast

SUBMITTER: Sew Peak-Chew

LAB HEAD: John S. O'Neill

PROVIDER: PXD044481 | Pride | 2023-09-22

REPOSITORIES: Pride

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Project description:Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained in the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the University of Tennessee Institutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embryos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (Â± 15 min.), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27Â± 1 ÂºC and 14:10h light:dark photoperiod. Experiment Overall Design: At 72 h post-fertilization, zebrafish larvae were exposed to lyophilized Microcystis and purified MC-LR at concentrations of 100 and 1,000 Âµg/L. Controls consisted of zebrafish system water (negative control) and zebrafish system water containing 0.05% ethanol (vehicle control). Larvae from both control groups as well as 100 Âµg/L MC-LR, 1,000 Âµg/L MC-LR, and lyophilized Microcystis were exposed in groups of 50 with three replicates and were sacrificed after 96 hours for total RNA extraction and subsequent microarray analysis. All larvae were exposed in beakers containing 100 ml of solution. Experiment Overall Design: Water samples for microcystin analysis and water quality measurements were taken during the experiment, and mortality and behavioral observations were recorded at 24-hour intervals. Microcystin analysis was conducted by protein phosphatase inhibition assay. Measured concentrations of microcystin-LR were 140 Â± 12 SD (low concentration) and 1,703 Â± 71 SD (high concentration). The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 Âµg/L. Water quality parameters measured included dissolved oxygen (6.7 mg/L), pH (6.9), total alkalinity (36 mg/L as CaCO3), total hardness (18 mg/L as CaCO3), and ammonia (<0.2 mg/L). No significant mortality or behavioral changes in larvae were observed during the exposure.

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Macromolecular condensation buffers intracellular water potential

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