Why cytoplasm is a colloid

The principal conclusions of the review were that the aqueous phase 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, as well as by unexpected binding interactions.

The purpose of this perspective is to review developments in the literature since and place them in context for the readership of Molecular Biology of the Cell. The high concentration of macromolecules and the extensive surface area presented by intracellular membranes in eukaryotic cells has led to proposals that association of intracellular water with surfaces leads to significant effects on its mobility and solvent properties compared with bulk water.

If true, this would profoundly affect our understanding of such fundamental cellular processes as 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. 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 different from what is found in bulk water or that either the rotational or the translational diffusion of solutes in cytoplasm is much affected by the anomalous viscosity of cellular water.

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 see Dix and Verkman, ; Zhou et al. Pure crowding effects typically are modeled as hard-sphere repulsive interactions that sterically exclude macromolecules in solution from the volume occupied by their neighbors.

According to this excluded-volume model, at high number concentration of macromolecules in solution, open space between molecules is reduced to the point that the free energy cost of making room for an additional molecule is thermodynamically significant.

In the absence of other attractive or repulsive interactions between the macromolecules, this free energy cost may promote intermolecular interactions that are energetically unfavorable in dilute solution, much as two people unknown to each other or with little in common may find themselves engaged in conversation at a crowded party. Excluded-volume effects may also stabilize the native conformation of ordered proteins by disfavoring more-extended conformations, much as large 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 as it takes longer to thread one's way around the other guests to cross the room at a crowded party.

In the extreme limit, macromolecular crowding might result in confinement of macromolecules within subvolumes of the cytoplasm for significant lengths of time, much as the press of other guests, furniture in the way. Many of the predicted effects 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. Fewer results are available for the effects of crowding on reaction kinetics, but a temperature-dependent increase in K cat has been reported for glucosephosphate dehydrogenase in well-defined crowded media Norris and Malys, It is now clear, however, that in the more complex intracellular environment, entropic excluded-volume effects are likely to be counteracted by enthalpic contributions from uncharacterized weak attractive or repulsive forces, with results that are not predictable a priori Inomata et al.

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 effects on any particular component Long et al.

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 considering the dynamics of a specific macromolecule in the cytoplasm of a specific cell. Over the past decade, a concept called anomalous diffusion has been adopted from the realm of physics to describe the diffusion of macromolecules in cells.

Although subdiffusion is more often applied to cytoplasm, a recent theoretical treatment proposes that superdiffusion is more likely Goychuk, This is a very active area of research, modeling, and simulation that so far has generated more heat than light regarding whether intracellular diffusion is anomalous, what the value of its exponent is, and what the detailed mechanism might be. Experimental data from the various studies on diffusion 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 often are difficult to test experimentally.

A recent article by Saxton succinctly summarizes the state of play and calls for development of a set of reproducible standard samples as positive controls that could be used to exclude the contributions of differing experimental conditions, methodologies, and artifacts to the experimental data, as well as to test the predictions of various mechanistic models that have been proposed.

Although anomalous diffusion clearly has implications for understanding any cellular process that depends on sampling of the cytoplasmic volume by diffusive transport, it is difficult to predict its effects without a clearer understanding 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 scale under consideration.

The idea of aqueous phase separation as a self-organizing force in the cell interior dates back to the father of modern cell biology, E. Wilson, who proposed that non—membrane-bound compartments such as P-granules and Cajal bodies could be explained by the principles of colloid chemistry Wilson, A colloid is a liquid with two phases: a microscopic droplet phase dispersed in a continuous phase.

Homogenized whole milk is the classic example. Since Wilson's time, the idea of phase separation as a mechanism for cellular microcompartmentation has gone in and out of vogue Welch and Clegg, 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. Phase 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, Other macromolecules and small solutes may partition into the droplet phase.

In crowded solutions with many different protein species, the total protein concentration in droplets is not necessarily higher than in the surrounding medium, and thus there may be no difference in refractive index to make them visible by microscopy.

Liquid droplets tend to adopt a minimum-energy, spherical shape unless deformed by external forces. They are dynamic in the sense that proteins readily exchange in and out of the droplet and that droplets 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, , as well as intracellular lipid droplets. Recent studies suggest that lipid droplets are not merely a trivial result of immiscibility between hydrophobic lipids and aqueous cytoplasm but instead may be the locus of lipid metabolism Walther and Farese, and also may serve as an intermediate compartment in the endoplasmic reticulum—associated protein degradation pathway Jo et al.

Overexpression of the protein interaction domains of two binding partners in the N-WASP signaling platform resulted in formation of similar liquid droplets in tissue culture cells Li et al. Further experimentation on living cells is required to decide whether and how these observations are relevant physiologically. An intriguing area of emerging research is the structure and function of bacterial microcompartments that encapsulate several enzymes of a metabolic pathway and sequester their substrates and intermediates Yeates et al.

These microcompartments have a highly organized icosahedral protein shell similar to virus capsids. Small pores in the walls of the shell are postulated to permit gated exchange of small molecules between the shell interior and the cytoplasm. No analogous structures have been reported for higher organisms, but several metabolic pathways have been reported to form supramolecular assemblies microscopically visible as foci or fibers O'Connell et al.

Regardless of the details of the physical chemistry of cytoplasm, certain general concepts are clear. Anything targeted to the cell surface by receptor specific ligands or on nanoparticles will enter the cell primarily by endocytosis, and their transport will reflect the behavior and fate of the endocytic vesicle containing them unless there is some mechanism of escape from the endocytic compartment.

Overexpressed proteins and agents delivered directly into the cytoplasm by methods that bypass the endocytic pathway will be subject to the same constraints on diffusion as endogenous intracellular solutes. It is inaccurate and misleading to think 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.

In the absence of binding, the rotational and translational mobility of small molecules, such as ions and small organic solutes, will be unaffected by crowding or by obstruction due to fixed obstacles and should reflect the viscosity of intracellular water, which current evidence suggests is essentially like bulk water.

Thus reaction rates that depend on diffusion of the reactants over short distances will be relatively unaffected by excluded-volume effects on diffusion and will approximate those measured in dilute solution. For macromolecule-sized solutes on longer time and distance scales, it is necessary to consider the possible effects of crowding, obstruction by fixed obstacles, and transient confinement on solute mobility. Predicting these from first principles is very difficult, 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 under study, as well as on the effects 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 any size may be slowed further. 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. The cytoplasmic compartment is inhomogeneous at nearly every length scale. In addition to randomly distributed local inhomogeneity driven stochastically by crowding and phase separation, nonrandom localization of intracellular vesicles, organelles, and supramolecular assemblies is a hallmark of eukaryotic cells.

It is becoming clear 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. It is important to remember that reported values for the physical properties of cytoplasm are spatially and temporally averaged and thus may not well describe the conditions in any particular subvolume of the cell. National Center for Biotechnology Information , U. Journal List Mol Biol Cell v.

So, we can grasp the fact that a cellular skeleton could be a valuable asset for the cell. But what makes this pipelike network truly robust is the fact that it can move. Cells can move, and some of them devote all their energy to this task. Cellular movement means locomotion for a unicellular being, which is useful when looking for resources, fleeing predators, etc.

When we look at cellular movement from a collective perspective, where many cells are tied together, other possibilities arise.

For example, many cells diminish their length in one specific axis—this makes muscle contraction possible. The same structural elements that constitute the cytoskeleton also allow some specialized cells to have additional abilities. The amoeboid-like movement of the plasma membrane is not the only means of locomotion that the tubular proteins allow for: some cells have tubular prolongations that can act as a whip and cause quick motion when they rhythmically beat.

For example, this is the case of sperm cells and some other eukaryotic and bacterial cells. In other cells, these prolongations can also act to thrust nutritious substances toward the cell to engulf them. While it is natural to think that cells are unorganized pools of gel that hold their internal structures, this is far from true. This permits the organelles to interact with one another and, in this way, perform highly coordinated and complex tasks.