The Effects of Macromolecular Crowding on Cell Physiology

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Biophysical Journal Volume 107 December 2014 2761–2766 2761 Biophysical Review

Connecting the Dots: The Effects of Macromolecular Crowding on Cell Physiology

Ma´rcio A. Moura˜o,1 Joe B. Hakim,2,3 and Santiago Schnell3,4,5,* 1Mathematical Biosciences Institute, Ohio State University, Columbus, Ohio; 2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland; 3Department of Molecular & Integrative Physiology, 4Department of Computational Medicine & Bioinformatics, and 5Brehm Center for Diabetes Research, University of Michigan Medical School, Ann Arbor, Michigan

ABSTRACT The physicochemical properties of cellular environments with a high macromolecular content have been system- atically characterized to explain differences observed in the diffusion coefficients, kinetics parameters, and thermodynamic properties of proteins inside and outside of cells. However, much less attention has been given to the effects of macromolecular crowding on cell physiology. Here, we review recent findings that shed some light on the role of crowding in various cellular processes, such as reduction of biochemical activities, structural reorganization of the cytoplasm, cytoplasm fluidity, and cellular dormancy. We conclude by presenting some unresolved problems that require the attention of biophysicists, biochemists, and cell physiologists. Although it is still underappreciated, macromolecular crowding plays a critical role in life as we know it.

Contrary to the typical in vitro media, the intracellular envi- Hoffmann et al. (23) extensively reviewed evidence ronment is densely packed with macromolecules, which showing that cell volume regulation is a byproduct of the occupy 5–40% of the total cellular volume (1). This packing regulation of cytosolic macromolecular crowding, ion is referred to as macromolecular crowding. The environ- pumps, and channels. Macromolecular crowding plays a ment is described as crowded and not highly concentrated role in the signaling of volume perturbation, as discussed because no single molecular species is necessarily present by Minton et al. (24) and Burg (25). Burg (25) proposed at a high density. The crowded environment contains that cell volume regulation is controlled by a crowding different molecules (proteins, nucleic acids, and/or polysac- sensor, i.e., a membrane-bound, two-state receptor. One charides) of various sizes and shapes. Importantly, crowding state would exclude volume and therefore be restricted by reduces the volume of solvent that is available for other crowding and trigger osmotic shrinkage. The other state molecules in the solution, which strongly affects molecules would do exactly the opposite. However, the link between with large molecular weights (2). This excluded-volume crowding and volume control remains relatively unexplored, effect has important consequences for cellular thermo- and a global control mechanism has not been identified dynamics and is reported to influence several cellular pro- beyond speculation. In this mini-review, we focus our atten- cesses (3–5). tion on another problem, which remains largely unexplored: The effects of macromolecular crowding on molecular the role of crowding in cell physiology and activity. What diffusion inside cells have been thoroughly examined over global, cell-level properties are controlled by macromolec- the past two decades. Experimental, theoretical, and compu- ular crowding? What are the physiological consequences tational studies have shown the existence of a crossover of overcrowding in the cell? Here, we address these from anomalous to regular diffusion in environments with questions by presenting recent findings that describe the a high macromolecular content (6–11). In the diffusion- relationship among macromolecular crowding, molecular limited regime, chemical reactions appear to be governed associations, cellular structure, intracellular signaling, and by fractal-like kinetics (12,13). Theoretical (14–16) and metabolism regulation in cell physiology. experimental (17–19) studies have found that enzyme-catalyzed reactions exhibit fractal-like kinetics in environments Macromolecular crowding decreases intracellular with a high macromolecular content (see Fig. 1). Macro- signaling and active protein transport molecular crowding can also increase the association rates of proteins (5) and protein-DNA interactions (20). This is The effects of macromolecular crowding in cell physiology consistent with the known effects of crowding in both can be investigated by exploiting the phenomenon of DNA replication (21) and protein folding (22). osmosis. Cells can be made to experience osmotic compression by adding higher solute concentrations in the extracel- lular medium. This volume contraction increases the overall Submitted August 25, 2014, and accepted for publication October 23, 2014. concentration of the macromolecular crowding agents *Correspondence: [email protected] in cells. In osmotically stressed bacteria, macromolecular Editor: Dennis Bray. Ó 2014 by the Biophysical Society 0006-3495/14/12/2761/6 $2.00 http://dx.doi.org/10.1016/j.bpj.2014.10.051 2762 Mouraõetal.

FIGURE 1 Macromolecular crowding affects diffusion and the rates of enzyme-catalyzed reactions. In low macromolecular crowding conditions (top), diffusion is classical and differs from the anomalous diffusion observed in more crowded environments (bottom). Under high crowding and

diffusion-limited conditions, the forward rate k1 is believed to follow fractal-like kinetics. Association

rate coefﬁcients, such as k1, decrease with increasing levels of crowding, but are also observed to increase under certain conditions. The formation of complexes is usually favored by high crowding. In the ﬁgure, S, E, C, and P represent the substrate, enzyme, enzyme-substrate complex, and product, respectively. The black squares represent the macromolecular crowding agents.

crowding decreases the diffusion coefficients of proteins is caused by an increase in the overall concentration of (10,11) and increases the association rates of protein-DNA crowding agents. This hypothesis is supported by measure- interactions (20). Since it is possible to measure and statis- ments of protein diffusion in the periplasm of Escherichia tically compare osmotic flux through signaling and meta- coli under osmotic stress (11). After sudden hyperosmotic bolic pathways, as well as other cellular functions, volume shock, the cytoplasm loses water as the periplasm gains contraction is widely used to study a variety of cellular pro- water. A net gain of water by the periplasm decreases its cesses (26). macromolecular crowding volume fraction. This leads to a In a recent article, Miermont et al. (27) reported a reduction 3-fold increase of the diffusion coefficient of a green fluo- in several biochemical processes, including cell signaling and rescent protein in the periplasm. These results are also protein transport in vesicles, as a consequence of a reduction supported by theoretical simulations that showed a decrease in cell volume. They found that a sudden reduction of yeast in diffusion coefficients (9,28) and reaction rates in an cell volume slows down the high-osmolarity glycerol increasingly crowded environment (4,14–16). (HOG) pathway required for osmotic adaptation. The strong What are the evolutionary consequences of the revers- reduction of cell volume leads to a decrease in the diffusion ibility of an osmotic-stress-mediated decrease in activity coefficient of Hog1p, as well as its phosphorylation rate of the aforementioned biological processes? One could and nuclear import. The inhibition of a volume regulation argue that single-celled organisms have evolved to use pathway when volume is strongly perturbed suggests a loss macromolecular agents to survive extreme osmotic stress in homeostatic control. It is possible, however, that the reduc- by entering dormancy. From the physicochemical point tion of the HOG pathway results from an extreme osmotic of view, the cytoplasm behaves like a colloidal suspen- stress induced by experimental conditions rather than by sion, which can undergo a glass transition by extreme the overall increase in the macromolecular crowding concen- crowding (29). tration. To further investigate this scenario, Miermont et al. (27) investigated the dynamics of other, unrelated biological Macromolecular crowding promotes molecular processes activated through different mechanisms. They associations and structural reorganization of the found that the dynamics of nuclear translocation of transcrip- cell tion factors (Yap1p, Crz1p, and Mig1p) is similarly reduced under osmotic stress. In addition, vesicular trafficking and Dormant cells can go through a structural reorganization endocytosis are also reduced under lower cellular volume. that makes them physically distinct from active cells. Interestingly, the reduction of these biochemical processes Recently, Petrovska et al. (30) investigated the organiza- is reversible, at least within a short timescale, through a reg- tional storage of proteins in dormant cells and found that ulatory volume increase (RVI) of the cells. the glutamine synthetase enzyme (Gln1) forms filaments Miermont et al. (27) hypothesized that the decrease in in yeast cells under low-nutrient conditions. Gln1 catalyzes protein diffusion and reaction rates by osmotic compression the ATP-dependent synthesis of glutamine from glutamate

Biophysical Journal 107(12) 2761–2766 Effects of Crowding on Cell Physiology 2763 and ammonium, but it is inactive in the filament form. is still ambiguous. Specifically, the rate and direction of In yeast cells, starvation causes the intracellular pH to volume control generally depend on the cell type, the decrease, which changes the charge state of protein surfaces transporter isoform, and changes in ATP conditions in and promotes protein association. However, the self-assem- ways that are not completely understood, warranting further bly of Gln1 into filaments only occurs in the presence of investigation. an environment with a high macromolecular crowding con- If a drop in intracellular pH does generally lead to RVD, tent. Interestingly, Petrovska et al. (30) also found other the increase in crowding agent concentration due to cellular metabolic enzymes assembling into filaments under acidic shrinkage may be sufficient to lead to the filament formation conditions. reported by Petrovska et al. (30). However, given the incon- Petrovska et al. (30) proposed that the structural reorgani- sistent effects of starvation-induced acidification on volume, zation of proteins into filaments is a mechanism to inactivate we cannot definitively ascertain that RVD is the source of and store key metabolic enzymes in states of stress, such as the filament formation in yeast cells. Petrovska et al. (30) cellular starvation. Sagot et al. (31) reported that under star- offer an alternate link between acidification and the assem- vation conditions, yeast cells disassemble their cytoskeleton bly of filaments. They suggest that low cytosolic pH may of actin filaments and reorganize it into actin bodies. After alter the charge distributions at the interfaces of protein sub- the cells are refed with a glucose-rich medium, actin bodies units, thereby decreasing intrinsic repulsion, or, alterna- disappear and actin filaments reassemble to form a cytoskel- tively, that the protons allosterically induce structural eton. The self-assembly of actin bodies could be driven by changes in the subunits. The molecular crowding itself macromolecular crowding, as it is well known that crowding would not be amplified, but the effects of crowding would increases such molecular association rates and accelerates be exacerbated in interactions with more readily self-assem- protein fibrillation (5). Remarkably, macromolecular crowd- bling structures. Although the details regarding the role of ing also plays a role in the self-organization of large crowding remain unknown, the regulation of cellular vol- molecular complexes that form nuclear structures, such as ume may serve as an evolutionary adaptation mechanism the nucleolus and other intranuclear bodies (32,33). for cell protection by processes driven by macromolecular Miermont et al. (27) and Petrovska et al. (30) propose crowding. During RVI, the overall macromolecular crowd- that macromolecular crowding plays a fundamental role ing concentration is low and the cell becomes sensitive to in cellular adaption to stressful environmental conditions. environmental perturbation. During RVD, the overall Although Petrovska et al. (30) did not show that cell volume crowding concentration is high and the cell is protected decreases with cytoplasmic acidification, there is evidence against environmental perturbations. that Ehrlich ascites tumor cells undergoing a regulatory vol- A point we have not considered yet is that enzyme inac- ume decrease (RVD) have low cytoplasmic pH (34). tivation triggered by starvation or lower pH causes cells to Mechanistically, the link between pH and volume regula- enter into a low metabolic state, similar to what is observed tion has been observed in various cell types, including most in hibernating animals. This effect may be driven by macro- mammalian cells and tissues, and involves several different molecular crowding, which clusters enzymes into filaments. transporter mechanisms (23). One class consists of the Naþ/ When clustered into filaments, enzymes are inactive. Is this Hþ exchangers, which electroneutrally expel cytosolic Hþ inactivation a mechanism for a reduction in cellular meta- in response to intracellular acidification (35). This is bolism? If this is the case, what are the physicochemical coupled with Cl/HCO3 exchange, which jointly imports and physiological implications of a metabolic reduction? NaCl (causing RVI) and reduces cytosolic Hþ concentration. This would imply that reduced pH, such as in starving Macromolecular crowding decreases metabolic conditions, would yield RVI, but ATP depletion inhibits this processes and cytoplasm fluidity exchange by limiting PIP-2 binding to the transporter (36). This Hþ efflux generally serves to counteract the passive In a recent article, Parry et al. (39) found that small particles acidifying influx of Hþ and HCO3, but is inhibited during (i.e., proteins) diffuse freely in the bacterial cytoplasm, conditions of starvation. Additionally, RVD is induced at whereas large particles (i.e., macromolecular complexes) low pH due to anion-dependent Kþ efflux (37), whereas exhibit anomalous diffusion. The diffusion of smaller parti- the overall rate of RVD is decreased in more acidic condi- cles is typical of that found in a liquid phase, and the larger tions. Several cation-Cl cotransporters (CCCs) that have particles display the anomalous diffusion found in heteroge- been implicated in RVD are inhibited at pH < 7.5 (KCC1, neous glass-like environments. Under 2,4-dinitrophenol- KCC3, and others), whereas KCC4 is activated. However, induced starvation, small particles were also restricted in KCCCs (and NKCCs) have the ability to import NH4þ, their diffusive motion, but less so than with larger species. probably on the Kþ-binding site, which leads to cytoplasmic The authors discovered this by tracking the movement of acidification (38). fluorescently labeled particles in the bacterium Caulobacter From this information, we can see that the true link be- crescentus and E. coli. Parry et al. (39) used volume exclu- tween cytosolic acidification and regulatory volume control sion (40) to explain this phenomenon, since the anomalous

Biophysical Journal 107(12) 2761–2766 2764 Mouraõetal. diffusion in heterogeneous glass-like environments is driven non was also observed by Parry et al. (39). The most rele- by physical crowding. The size dependency of diffusion vant observation made by Guo et al. (42) is that there is a restriction is compatible with the size dependency of the correlation between the overall activity of a cell and the volume-exclusion effect in that larger particles have less active force fluctuations of protein complexes and organ- available volume than smaller particles in a similarly elles in the cell. Transporting and mixing ribosomes may crowded environment (3) and thus are more strongly influ- increase the probability that ribosomes will encounter pro- enced by crowding. teins, and the removal of enzyme products for the synthesis Remarkably, Parry et al. (39) discovered that the diffusion sites would increase enzymatic activity. As the ensemble of small particles can become anomalous in dormant bacte- force increases in a cell, there is more mixing and transport ria, whereas anomalous diffusion is suppressed by metabolic of cellular components. As an example, Guo et al. (42) use activity in a particle-size-dependent manner. They found the human breast cancer cell line MCF-7. This cell line has that metabolic activity fluidizes the cytoplasm to enhance enhanced metabolic and proliferative rates, and exhibits a molecular motion. How is it possible that a passive physico- higher force spectrum. As cells are considerably confined chemical process can change with the metabolic state of the by macromolecular crowding, there is a decrease in the cell? A multitude of simultaneously occurring cellular pro- overall transport and metabolic activity. cesses could play a role in enhancing the diffusion observed in metabolically active cells. Based on the theory of Spitzer Conclusions (41), Parry et al. (39) hypothesized that metabolic activity continuously changes the structural configuration of the Macromolecular crowding is gaining recognition as a major cytoplasm by altering the hydrophobicity and electrostatic player in intracellular biochemical interactions. Although interactions of molecules with macromolecular crowding. its effect on diffusion and reaction kinetics has been exten- We have an alternative theory for the fluidization of the sively addressed, very little is known about how macromo- cytoplasm by metabolic activity. In dormant cells, the meta- lecular crowding affects cell physiology. The cytoplasm is bolic activity is suppressed and the cell enters into an ATP- densely packed with macromolecules, and the function of depleted state. Under these conditions, the cells experience many of these macromolecules remains unknown. All cells a RVD because the active transport pumps responsible for have evolved to synthesize crowding agents, which are used maintaining the homeostatic cell volume are inactive. This to regulate diffusion, reaction rates, biochemical processes, will inevitably lead to an overall increase in the macromo- and cellular organization. Remarkably, cells have evolved lecular crowding concentration that hinders diffusion. adaptation and survival strategies to enter and exit dormancy Once the metabolic activity builds up, the cell will increase by changing the physicochemical properties of the cyto- its ATP concentration and activate the ATP-dependent trans- plasm via macromolecular crowding. Macromolecular port pumps responsible for volume regulation. The cell will crowding plays a critical role during the RVD, when the then go through a RVI phase, decreasing the overall macro- overall concentration of macromolecular crowding is high molecular crowding concentration and increasing molecular and cells undergo a glass transition. Based on the findings diffusion. reviewed in this article, we propose that macromolecular In a recent study, Guo et al. (42) provided additional clues crowding affects cellular physiological processes through regarding the physiological role of macromolecular crowd- cellular volume regulation (see Fig. 2). Cells change be- ing by investigating the movement of larger intracellular tween dormant and active states, thereby changing biochem- components. They introduced a technique called force spec- ical activity and other processes by RVD and RVI. trum microscopy, which they used to measure the ensemble The most remarkable findings recently made by Mier- forces due to the overall activity of the cell. The authors mont et al. (27), Petrovska et al. (30), and Parry et al. demonstrated that the ensemble forces are due in part to (39), as discussed in this article, are mostly limited to pro- active processes and can be reduced by ATP depletion karyotic single-cell organisms. It remains to be investigated in the absence of Myosin II activity. Interestingly, the whether similar behaviors are found in single-cell organisms ensemble force reduction scales with the size of the tracer lacking cell walls and in multicellular organisms. Many particles; that is, thermal-driven motion is more significant other open questions and challenges concerning this topic for smaller particles, but not as effective at moving the exist as well. Recently, it was shown that macromolecular tracers (100–500 nm), mitochondria, vesicles, or protein crowding modulates the dynamics of gene expression in complexes, which are transported via active motors. This artificial cells (43). Theoretical work (44,45) suggests that is consistent with the size selectivity of the slowing effect macromolecular crowding serves as a modulator of gene of crowding on intracellular components. Guo et al. (42) expression. The effects of macromolecular crowding as also attribute the motion of individual proteins (Dendra2) regulators of gene expression remain to be explored system- to active transport, but, as we pointed out, ATP depletion atically in living cells. Can we develop experimental tech- indirectly halts small-particle transport and the cytoplasm niques to measure macromolecular crowding inside the becomes glassy under starvation conditions. This phenome- cells? A new type of ratiometric fluorescent molecular rotor

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Dormant FIGURE 2 Modulation of cellular volume to control physiological processes via macromolec- Regulatory Volume Decrease ular crowding. The active cell and the dormant cell interchange states by undergoing RVD or RVI, respectively. In the dormant cells, the higher concentration of crowding agents (black dots in cytoplasm) leads to the formation of protein com- Regulatory Volume Increase plexes. Proteins are indicated by light blue dots, and complexes represent ﬁlaments and clusters of proteins. In addition, the chromatin is compressed by higher crowding. Note that the quantity of crowding agents does not necessarily change, but a reduction of volume leads to a higher overall concentration of crowding agents. Upon formation of _ Volume + protein ﬁlaments and clusters in response to volume contraction, metabolic activity is slowed, + Protein-Protein Association _ which may correspond to a depletion of nutrients stopping active transport and inhibiting RVI, thus _ Cytoplasm Fluidization + making the cell dormant.

has been developed to quantify and image intracellular vis- In short, we have a complex regulatory web of inter- cosity in live cells (46). If this type of technology can be actions describing fundamental cellular processes, and used to measure the overall concentration of macromolec- perhaps the best way to elucidate this is to apply both exper- ular crowding, we may be able to resolve several open ques- imental and theoretical modeling studies. The former would tions. Is there a certain crowding concentration at which the establish evidence of the interactions as demonstrated here, cytoplasm behaves differently from a simple viscous fluid? and the latter would provide a means of investigating the What are the mechanisms responsible for the regulation of nonlinearity and ambiguous feedback interactions mecha- macromolecular crowding synthesis? How is the regulation nistically. In addition, analysis of this complex regulatory of cell volume linked to macromolecular crowding? Is this network requires the integration of information across regulation driven by signaling or metabolism? Lastly, how spatial, temporal, and functional scales. With the advent are signaling and metabolism linked to active pumps and of powerful computing platforms and systems biology, the channels to regulate cell volume? development of multiscale models may enable researchers To illustrate our limited understanding of the mechanisms to comprehensively investigate the effects of macromolec- that regulate the effects of macromolecular crowding on cell ular crowding on cell physiology by testing diverse mecha- physiology, we can holistically look at how the recent find- nisms and selecting those that best explain the experimental ings discussed above may tie together. The big picture estab- observations. In the meantime, there is only one certainty: lished by combining these findings is a complicated web of although macromolecular crowding has the potential to regulatory interactions. This creates a confusing picture and radically alter our understanding of the cellular environ- leaves us with a chicken-and-egg type problem about how ment, our current scientific knowledge about macromolec- volume regulation, protein-protein associations, cytoplasm ular crowding is still in its infancy. fluidization, and biochemical activity are tied together. We can say that crowding affects metabolic activity through We thank Michael Vincent (University of Michigan) for critically reading the manuscript. filament storage of enzymes and metabolites, but does metabolism downregulate crowding by inhibiting volume or MAM was supported by a Mathematical Bioscience Institute postdoctoral fellowship funded through National Science Foundation (Grant No. changing the electrostatic composition of macromolecular DMS-0931642). This work was partially supported by the James S. crowding directly? We know that volume contraction in- McDonnell Foundation (Grant No. 220020223) under the 21st Century Sci- creases crowding content, but is crowding generally used ence Initiative Studying Complex Systems Program and the National Insti- as a homeostatic volume sensor in all cells? We could tute of Diabetes and Digestive Diseases (Grant No. R25 DK088752). have starvation leading to a volume decrease, inducing an overall increase in crowding concentration, and then re- stricting metabolism. Alternatively, we could have volume REFERENCES contraction due to low ATP availability, leading to an overall high crowding concentration, which in turn would slow the 1. Ellis, R. J. 2001. Macromolecular crowding: obvious but underappreci- metabolic processes until nutrients became available. ated. Trends Biochem. Sci. 26:597–604.

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