Microorganisms maintain crowding homeostasis

Berg, Jonas van den; Boersma, Arnold J.; Poolman, Bert

doi:10.1038/nrmicro.2017.17

Cited by 187 publications

(161 citation statements)

References 106 publications

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“…Because water primarily drives volume change, whereas macromolecular content remains constant, we account for the relative free volume change,ṽ f =ṽ −ṽ occ . The occupied relative volumẽ v occ in the cell, composed of all solutes that do not readily diffuse upon volume change, is estimated to be ∼30% in isosmotic conditions (37,38). In Fig.…”

Section: Resultsmentioning

confidence: 99%

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Sukenik

Ren

Gruebele

2017

Proc. Natl. Acad. Sci. U.S.A.

123

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Weakly bound protein complexes play a crucial role in metabolic, regulatory, and signaling pathways, due in part to the high tunability of their bound and unbound populations. This tunability makes weak binding (micromolar to millimolar dissociation constants) difficult to quantify under biologically relevant conditions. Here, we use rapid perturbation of cell volume to modulate the concentration of weakly bound protein complexes, allowing us to detect their dissociation constant and stoichiometry directly inside the cell. We control cell volume by modulating media osmotic pressure and observe the resulting complex association and dissociation by FRET microscopy. We quantitatively examine the interaction between GAPDH and PGK, two sequential enzymes in the glycolysis catalytic cycle. GAPDH and PGK have been shown to interact weakly, but the interaction has not been quantified in vivo. A quantitative model fits our experimental results with log K d = −9.7 ± 0.3 and a 2:1 prevalent stoichiometry of the GAPDH:PGK complex. Cellular volume perturbation is a widely applicable tool to detect transient protein interactions and other biomolecular interactions in situ. Our results also suggest that cells could use volume change (e.g., as occurs upon entry to mitosis) to regulate function by altering biomolecular complex concentrations.quinary interactions | protein-protein interactions | cell volume | FRET | live-cell microscopy F rom forming active enzymatic complexes to facilitating signal transduction in regulatory networks, protein interactions are pivotal to cell function. Strong interactions, with dissociation constants (K d ) of nanomolar and lower (1, 2), can be measured accurately both in vitro and in vivo (3, 4). These interactions are ideal in cases where the complex should form with high fidelity and persist. The downside of strong binding is that its regulation in the cellular environment is limited: Once a complex is formed, it seldom dissociates.Another type of protein complexes relies on weak, transient interactions, collectively termed quinary interactions (5-7). Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9). Their transient nature is key to their function: Unlike tightly bound complexes, quinary interactions are highly sensitive to variations in their environment and respond rapidly to changes in temperature, pressure, pH, or the local concentration of surrounding molecules. The sensitivity of quinary interactions makes them important in fine-tuning cellular processes. For example, it has been proposed that sequential metabolic enzymes could associate to improve substrate transfer between catalytic enzymes (10), that weak protein association can mediate cytokine release (11), or that phase-separated protein droplets held together by quinary interactions could serve for cellular storage or stress response (12, 13).Despite growing interest, quantification of quinary inte...

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Section: Resultsmentioning

confidence: 99%

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Sukenik

Ren

Gruebele

2017

Proc. Natl. Acad. Sci. U.S.A.

123

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“…Inhomogeneous distribution could potentially occur under, for example, starvation conditions, or when other stresses are imposed on the cell (50)(51)(52). In these cases, the probes may indicate changes in the superstructure of the cytoplasm, especially when combined with classical volume fraction determinations from cell dry weight and probe diffusion measurements (17,44).…”

Section: Discussionmentioning

confidence: 99%

“…The polyethylene glycol 10,000 (PEG)-based sensor may function via a similar mechanism to our sensor, whereas the mechanism behind the destabilization of the YFP sensor is not yet clear. Crowding can also be inferred from diffusion measurements, among other methods (17), but these are strongly dependent on other parameters such as confinement, viscosity, and nonspecific attractive interactions.…”

Section: Introductionmentioning

confidence: 99%

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Zhang

Åberg

Eerden

et al. 2017

Biophysical Journal

Self Cite

100

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Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Fö rster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia coli are more complicated, where FRETincreases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecular volume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.

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“…Procedures have been developed to incorporate integral membrane proteins or lipid-anchored proteins into the membrane and to include enzymes and small molecules into the vesicle lumen. [59] Increasing the outside osmolality causest he vesicles to shrink due to water efflux, and the luminalc ontentsa re concentrated. [55] We have produced submicron and micrometre-size proteoliposomes with up to 50 mg mL À1 of protein or cell lysate in the vesicle lumen, [58] but it is technically challengingt oa chieve in vivo-like crowding levels (200-300 mg mL À1 ).…”

Section: Vesicle Systemsmentioning

confidence: 99%

“…[55] We have produced submicron and micrometre-size proteoliposomes with up to 50 mg mL À1 of protein or cell lysate in the vesicle lumen, [58] but it is technically challengingt oa chieve in vivo-like crowding levels (200-300 mg mL À1 ). [59] Increasing the outside osmolality causest he vesicles to shrink due to water efflux, and the luminalc ontentsa re concentrated. The shrinking of the vesiclesi sr eversible;i to ccurs when osmolytes are taken up or the outside osmolality is reduced.…”

Section: Vesicle Systemsmentioning

confidence: 99%

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

et al. 2019

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We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from molecular components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochemical ion gradients. We have estimated the demand for ATP by polymer synthesis and maintenance processes in small cell‐like systems, and we describe circuits to control the need for ATP. We also present fluorescence‐based sensors for pH, ionic strength, excluded volume, ATP/ADP, and viscosity, which allow the major physicochemical conditions inside cells to be monitored and tuned.

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Microorganisms maintain crowding homeostasis

Cited by 187 publications

References 106 publications

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

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