What Is The Chemical Makeup Of Cytoplasm

• Journal List
• Mol Biol Prison cell
• v.24(17); 2013 Sep 1
• PMC3756912

Mol Biol Prison cell. 2013 Sep i; 24(17): 2593–2596.

The physical chemistry of cytoplasm and its influence on cell role: an update

William Bement, Monitoring Editor

Academy of Wisconsin

Received 2013 Apr 2; Revised 2013 May 8; Accepted 2013 Jul 2.

Abstract

From the point of view of intermolecular interactions, the cytoplasmic space is more like a crowded party in a house full of furniture than a game of tag in an empty field. Understanding the physical chemical properties of cytoplasm is thus of key importance for understanding cellular function. This article attempts to provide an entrée into the electric current literature on this subject area and offers some general guidelines for thinking about intracellular biochemistry.

INTRODUCTION

Cellular cytoplasm is the context for all intracellular activities that are not sequestered within membrane-bounded organelles, and thus its concrete chemic backdrop influence cardinal cellular functions, including poly peptide folding, enzyme catalysis, intracellular signaling, intracellular transport, and localization of molecules and organelles, likewise as the fate of nanoparticles and therapeutic agents targeted to cells. In 2000, I published a review article in which I attempted to summarize the extensive literature begetting on the nature of the prison cell interior and in particular the extent to which it departs from the platonic dilute solution often causeless in classical biochemistry (Luby-Phelps, 2000 ). The principal conclusions of the review were that the aqueous stage of the cytoplasm is not a bag of freely diffusing enzymes but is crowded with macromolecules and that diffusive transport and partitioning of macromolecules and organelles in cytoplasm is highly restricted by steric hindrance, equally well every bit by unexpected bounden interactions. The purpose of this perspective is to review developments in the literature since 2000 and place them in context for the readership of Molecular Biology of the Cell.

INTRACELLULAR WATER

The high concentration of macromolecules and the extensive surface surface area presented by intracellular membranes in eukaryotic cells has led to proposals that association of intracellular h2o with surfaces leads to significant effects on its mobility and solvent properties compared with majority h2o. If true, this would profoundly touch on our understanding of such fundamental cellular processes every bit diffusion-limited biochemical interactions and protein folding. Experimental support for this view at the time of the previous review lacked direct measurement of water mobility in intact cells under physiological conditions. Since then, such measurements have become available (Jasnin et al., 2008 ; Persson and Halle, 2008 ; Stadler et al., 2008 ), including a study of water relaxation times in cubic micro­meter–sized subvolumes inside living COS-1 cells (Potma et al., 2001 ). These studies suggest that at nearly 10–15% of intracellular water has contradistinct mobility, and that although h2o molecules in the outset layer of hydration may have relaxation times 10- to 15-fold lower than majority, this does not propagate to water molecules over any significant altitude, as the measured overall viscosity of intracellular water is merely seventy% college than that of majority water. Furthermore, water molecules hydrating proteins and other surfaces appear to be readily exchangeable with the bulk. Based on the evidence, there is no reason to suppose that hydration and solvation in the cell cytoplasm are significantly dissimilar from what is found in bulk water or that either the rotational or the translational diffusion of solutes in cytoplasm is much afflicted by the anomalous viscosity of cellular h2o.

WHAT'Due south IN A Crowd?

The recognition that the cell cytoplasm is a highly crowded medium has led to much study and theorizing about the effects of macromolecular crowding on cellular biochemistry (for reviews run across Dix and Verkman, 2008 ; Zhou et al., 2008 ). Pure crowding furnishings typically are modeled as hard-sphere repulsive interactions that sterically exclude macromolecules in solution from the volume occupied by their neighbors. Co-ordinate to this excluded-book model, at high number concentration of macromolecules in solution, open space betwixt molecules is reduced to the point that the free energy price of making room for an boosted molecule is thermodynamically significant. In the absence of other bonny or repulsive interactions betwixt the macromolecules, this free energy toll may promote intermolecular interactions that are energetically unfavorable in dilute solution, much as two people unknown to each other or with niggling in common may find themselves engaged in conversation at a crowded party. Excluded-volume effects may besides stabilize the native conformation of ordered proteins past disfavoring more than-extended conformations, much as big arm movements are restricted at a crowded party for fear of hitting other guests. In addition, the crowding molecules present obstacles that may retard long-range translational movement, much equally it takes longer to thread one's fashion around the other guests to cantankerous the room at a crowded party. In the extreme limit, macromolecular crowding might issue in confinement of macromolecules within subvolumes of the cytoplasm for significant lengths of time, much equally the press of other guests, furniture in the style. and a narrow doorway may temporarily forestall one from moving from one room to some other at the crowded house party.

Many of the predicted furnishings of macromolecular crowding have been demonstrated to occur in vitro in well-defined model systems. Several studies have shown that macromolecular crowding can promote protein folding (e.g., Hong and Gierasch, 2010 ; Stagg et al., 2011 ) and stabilize the meaty conformation of isolated metaphase chromatin (Hancock, 2012 ). Fewer results are bachelor for the effects of crowding on reaction kinetics, but a temperature-dependent increase in Yard _{true cat} has been reported for glucose-6-phosphate dehydrogenase in well-defined crowded media (Norris and Malys, 2011 ). Information technology is now articulate, however, that in the more complex intracellular surround, entropic excluded-volume effects are likely to be counteracted by enthalpic contributions from uncharacterized weak attractive or repulsive forces, with results that are not anticipated a priori (Inomata et al., 2009 ; Elcock, 2010 ; Schlesinger et al., 2011 ; Wang et al., 2012 ; Zhang et al., 2012 ). An additional complication is that in complex mixtures like cytoplasm, crowded with multiple species of macromolecules of differing size, shape, and flexibility, some species may spontaneously demix and condense into stable droplet phases dispersed in the bulk, with unpredictable furnishings on any particular component (Long et al., 2005 ). Thus it now seems that bottom-up approaches such as experiments in well-defined model systems in vitro or simulations in silico will provide only very general insight when because the dynamics of a specific macromolecule in the cytoplasm of a specific prison cell.

Anomalous Diffusion (SUB OR SUPER?)

A variety of experimental measurements suggest that long-range translational improvidence of macromolecules in the cytoplasm may non match the expectations of normal diffusion in dilute aqueous solution, for which mean-squared displacement (MSD) is a linear function of elapsed fourth dimension (MSD ∝ t^x , where x = 1). Over the by decade, a concept called dissonant diffusion has been adopted from the realm of physics to describe the diffusion of macromolecules in cells. In anomalous diffusion the human relationship of MSD with time is nonlinear: cases in which the measured diffusion coefficient appears to decrease with elapsed time are referred to as subdiffusion (ten < i), whereas cases in which the apparent diffusion coefficient increases with elapsed time are referred to equally superdiffusion (x > 1). Although subdiffusion is more frequently applied to cytoplasm, a recent theoretical treatment proposes that superdiffusion is more than probable (Goychuk, 2012 ). This is a very agile area of research, modeling, and simulation that so far has generated more estrus than light regarding whether intracellular diffusion is dissonant, what the value of its exponent is, and what the detailed machinery might exist. Experimental data from the various studies on improvidence in living cells are difficult to reconcile due to the nonoverlapping time and spatial scales of different methods of measurement, and the conclusions drawn from models and simulations oft are hard to test experimentally. A recent article by Saxton (2012 ) succinctly summarizes the country of play and calls for development of a set of reproducible standard samples as positive controls that could exist used to exclude the contributions of differing experimental conditions, methodologies, and artifacts to the experimental information, also every bit to test the predictions of various mechanistic models that have been proposed. Although anomalous diffusion clearly has implications for understanding whatever cellular process that depends on sampling of the cytoplasmic volume by diffusive send, it is difficult to predict its effects without a clearer agreement of the extent to which anomalous diffusion actually describes intracellular dynamics. Reaction kinetics may be either faster or slower, depending on the type of anomalous diffusion and the time and distance calibration under consideration.

PHASE SEPARATION AND MICROCOMPARTMENTATION

The idea of aqueous stage separation equally a cocky-organizing force in the prison cell interior dates back to the father of modernistic prison cell biological science, E. B. Wilson, who proposed that not–membrane-bound compartments such as P-granules and Cajal bodies could exist explained by the principles of colloid chemistry (Wilson, 1899 ). A colloid is a liquid with two phases: a microscopic droplet stage dispersed in a continuous phase. Homogenized whole milk is the classic example. Since Wilson's time, the thought of phase separation equally a mechanism for cellular microcompartmentation has gone in and out of vogue (Welch and Clegg, 2010 ). Currently its popularity is resurging, partly as a result of renewed appreciation for how crowded the cytoplasm is. Crowding-induced phase separation is a well-studied phenomenon in colloid science. Stage separation of immiscible proteins in a crowded solution typically leads to formation of liquid droplets enriched in one or a subset of interacting proteins (Weber and Brangwynne, 2012 ). Other macromolecules and small-scale solutes may partition into the droplet phase. In crowded solutions with many different poly peptide species, the total protein concentration in aerosol is not necessarily higher than in the surrounding medium, and thus there may be no difference in refractive alphabetize to make them visible by microscopy. Liquid droplets tend to adopt a minimum-energy, spherical shape unless deformed past external forces. They are dynamic in the sense that proteins readily exchange in and out of the droplet and that aerosol encountering each other may coalesce. Examples of well-known intracellular inclusions that exhibit droplet behavior include P-bodies in germline cells of Caenorhabditis elegans and Cajal bodies in the nucleus (Hyman and Simons, 2012 ), as well as intracellular lipid droplets. Recent studies advise that lipid droplets are not merely a trivial result of immiscibility between hydrophobic lipids and aqueous cytoplasm merely instead may be the locus of lipid metabolism (Walther and Farese, 2012 ) and likewise may serve as an intermediate compartment in the endoplasmic reticulum–associated protein deposition pathway (Jo et al., 2013 ).

Recent reports show that purified components of the Due north-WASP signaling pathway (Li et al., 2012 ) and RNA-binding proteins in a jail cell lysate (Kato et al., 2012 ) spontaneously phase separate under certain conditions in vitro. Overexpression of the protein interaction domains of two bounden partners in the Due north-WASP signaling platform resulted in formation of similar liquid droplets in tissue culture cells (Li et al., 2012 ). In these studies, phase separation was establish to depend on multivalent weak interactions between low-complexity repeat domains and/or disordered hydrophobic domains. Farther experimentation on living cells is required to decide whether and how these observations are relevant physiologically.

An intriguing area of emerging research is the construction and function of bacterial microcompartments that encapsulate several enzymes of a metabolic pathway and sequester their substrates and intermediates (Yeates et al., 2011 ). These microcompartments have a highly organized icosahedral protein shell similar to virus capsids. Pocket-sized pores in the walls of the shell are postulated to allow gated exchange of pocket-sized molecules betwixt the vanquish interior and the cytoplasm. No analogous structures take been reported for higher organisms, but several metabolic pathways accept been reported to grade supramolecular assemblies microscopically visible as foci or fibers (O'Connell et al., 2012 ).

SIZE MATTERS AND THINGS Modify WITH Fourth dimension

Regardless of the details of the physical chemistry of cytoplasm, certain full general concepts are clear. Anything targeted to the jail cell surface past receptor specific ligands or on nanoparticles will enter the prison cell primarily by endocytosis, and their send will reflect the behavior and fate of the endocytic vesicle containing them unless at that place is some mechanism of escape from the endocytic compartment. Overexpressed proteins and agents delivered directly into the cytoplasm by methods that featherbed the endocytic pathway volition exist subject to the same constraints on improvidence equally endogenous intracellular solutes. It is inaccurate and misleading to retrieve of cytoplasm as a homogeneous medium like a dilute solution, with a single viscosity that characterizes the rotational mobility of small molecules, the long-range translational diffusion of solutes, and the consistency of the bulk. The observed mobility of solutes in crowded, circuitous mixtures such as the jail cell interior volition depend on the size of the solute and the time/space interval over which it is observed. In the absence of binding, the rotational and translational mobility of small molecules, such as ions and small organic solutes, will be unaffected past crowding or by obstruction due to stock-still obstacles and should reflect the viscosity of intracellular water, which current testify suggests is substantially like majority water. Even macromolecules the size of a typical globular protein (∼three nm in radius) may diffuse normally over extremely brusque distances or on very brusque fourth dimension scales because the probability of encountering barriers to improvidence in this space-fourth dimension regime is relatively depression. Thus reaction rates that depend on diffusion of the reactants over short distances volition be relatively unaffected by excluded-volume effects on diffusion and will estimate those measured in dilute solution. For macromolecule-sized solutes on longer fourth dimension and altitude scales, it is necessary to consider the possible effects of crowding, obstacle by fixed obstacles, and transient confinement on solute mobility. Predicting these from first principles is very hard, if not impossible, and for real biological molecules in the cytoplasm of living cells additionally depend on the specific size, shape, and deformability of the molecule nether study, as well equally on the furnishings of weak attractive or repulsive forces. To the extent that they experience transient binding interactions or partition into droplet phases, the mobility of molecules of whatever size may be slowed farther. In this regard, two recent studies indicate that binding interactions are the dominant factors responsible for the extremely low mobility of globular proteins observed in Escherichia coli (Nenninger et al., 2010 ; Wang et al., 2011 ).

LOCATION, LOCATION, LOCATION

The cytoplasmic compartment is inhomogeneous at nearly every length scale. In addition to randomly distributed local inhomogeneity driven stochastically past crowding and stage separation, nonrandom localization of intracellular vesicles, organelles, and supramolecular assemblies is a authentication of eukaryotic cells. Information technology is becoming articulate that in prokaryotes, as well as in eukaryotes, individual protein and RNA molecules may also be nonrandomly localized within the cytoplasmic compartment (Nevo-Dinur et al., 2012 ). An extensive literature suggests that the concentrations of even pocket-size signaling molecules such as army camp and Ca²⁺ may be locally regulated. It is important to remember that reported values for the physical properties of cytoplasm are spatially and temporally averaged and thus may not well depict the atmospheric condition in whatsoever particular subvolume of the cell.

Abridgement used:

MSD	hateful squared deportation

Footnotes

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