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Cytosol (also termed the intracellular fluid or cytoplasmic matrix) is the continuous, aqueous liquid phase of the interior of a living cell that remains after excluding the cell nucleus and all membrane-bound organelles (such as mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and vacuoles). Introduced as an operational biochemical term by Henry A. Lardy in 1965 to describe the soluble supernatant fraction obtained after ultracentrifugation of lysed cells at 100,000 × g, the intact in vivo cytosol occupies approximately 50% to 70% of total cell volume in typical eukaryotes and nearly the entirety of the intracellular volume in prokaryotes. Far from being a dilute, passive salt solution, the cytosol is a viscoelastic, macromolecularly crowded colloidal matrix permeated by the cytoskeleton and organized into dynamic, membrane-less biomolecular condensates that orchestrate glycolysis, ribosomal protein translation, and intracellular signal transduction.

In rigorous cell biology, cytoplasm and cytosol are not synonymous:

• Cytoplasm encompasses the entire contents of a cell enclosed within the plasma cell membrane outside the nucleus—including both the aqueous cytosol and all suspended cytoplasmic organelles (mitochondria, plastids, peroxisomes, lysosomes, endosomes, and the endomembrane network).
• Cytosol refers strictly to the aqueous phase and its dissolved ions, small metabolites, soluble enzymes, ribosomes, proteasomes, and cytoskeletal filaments outside those organelle membranes.

A defining biophysical property of all living organisms is the sharp electrochemical disequilibrium maintained across the plasma membrane between the cytosol and the extracellular fluid (or blood plasma in metazoans). Driven continuously by the Na⁺/K⁺-ATPase pump, plasma membrane Ca²⁺-ATPases (PMCA), and Na⁺/H⁺ exchangers—which consume 20% to 40% of a resting mammalian cell's adenosine triphosphate (ATP) budget—the cytosol preserves an ancient marine-like ionic profile enriched in potassium and magnesium while rigorously excluding sodium, chloride, and free calcium:

The 10,000-Fold Calcium Gradient as a Universal Signaling Switch

Because high concentrations of free Ca²⁺ ions precipitate inorganic phosphate (HPO₄²⁻)—which is abundant at 1–10 mM in the cytosol for ATP synthesis— into insoluble hydroxyapatite crystals, early cells evolved powerful pumps to keep resting cytosolic free Ca²⁺ below 100 nanomolar (10⁻⁷ M). Most intracellular calcium is sequestered inside the lumen of the smooth endoplasmic reticulum (or sarcoplasmic reticulum in muscle) via SERCA pumps, or buffered by cytosolic calcium-binding proteins such as calmodulin. Consequently, the transient opening of voltage-gated or IP₃-gated Ca²⁺ channels floods the cytosol with a 10- to 100-fold relative surge of Ca²⁺ within milliseconds, triggering muscle contraction, fertilization, and synaptic vesicle exocytosis.

Although water constitutes approximately 70% of the cytosol by weight, textbook illustrations depicting enzymes floating in vast expanses of empty water are physically inaccurate.

The Excluded Volume Effect (Macromolecular Crowding)

The cytosol contains 200 to 300 mg/mL of dissolved proteins, RNA molecules, and polysaccharides, occupying 20% to 35% of the total volume. In this densely packed environment, the average surface-to-surface distance between adjacent macromolecules is only 1 to 2 nanometers—roughly the thickness of three to six layers of hydration water molecules. As analyzed by Allen Minton and R. John Ellis, this macromolecular crowding creates a powerful thermodynamic excluded volume effect:

• Translational diffusion of medium-sized globular proteins (e.g., 30–50 kDa) is slowed 3- to 5-fold relative to pure water (D_cytosol ≈ 10–30 μm²/s), while large complexes (>500 kDa) experience anomalous subdiffusion caged by the cytoskeleton.
• Thermodynamic activity coefficients increase by orders of magnitude, strongly shifting chemical equilibria toward compact folded protein states, oligomeric enzyme assembly (metabolons), and actin/microtubule polymerization because folded complexes occupy less steric volume and maximize the translational entropy of surrounding solvent molecules.

Liquid-Liquid Phase Separation (LLPS) and Biomolecular Condensates

Despite lacking internal lipid membranes, the cytosol compartmentalizes biochemistry into dynamic, micron-scale droplets known as biomolecular condensates or membrane-less organelles—discovered in 2009 by Clifford Brangwynne and Anthony Hyman through studies of Caenorhabditis elegans P granules. Driven by multivalent weak interactions among intrinsically disordered protein regions (IDRs) and RNA molecules, the cytosol undergoes liquid-liquid phase separation (LLPS) (analogous to oil droplets demixing from vinegar) to form:

• Stress granules and Processing bodies (P-bodies): Sequestering untranslated mRNAs and mRNA-decay machinery during cellular stress.
• Signaling puncta: Concentrating T-cell receptor kinases or innate immune sensors (such as cGAS-STING) to accelerate reaction kinetics by 10- to 100-fold.
• Glycolytic bodies (G-bodies): Transient condensates that co-localize glycolytic enzymes under hypoxic stress to channel substrates directly between active sites.

The cytosol serves as the primary reaction vessel for major catabolic, anabolic, and quality-control pathways in eukaryotic and prokaryotic cells:

1. Carbohydrate Metabolism: All ten reactions of glycolysis (converting glucose to pyruvate), the pentose phosphate pathway (generating NADPH for reductive biosynthesis and ribose-5-phosphate for nucleotides), gluconeogenesis (downstream of phosphoenolpyruvate carboxykinase), and glycogenesis/glycogenolysis (the synthesis and breakdown of branched glycogen granules) occur directly in the cytosol.
2. Lipid and Nucleotide Biosynthesis: De novo fatty acid synthesis (catalyzed by the massive homodimeric fatty acid synthase complex using acetyl-CoA and malonyl-CoA), the early mevalonate steps of cholesterol biosynthesis, and de novo purine and pyrimidine nucleotide synthesis take place in the cytosolic phase.
3. Protein Translation, Folding, and Proteasomal Degradation: Free cytosolic ribosomes (80S in eukaryotes, 70S in prokaryotes) translate mRNAs encoding all cytosolic, nuclear, mitochondrial, chloroplast, and peroxisomal proteins. Newly synthesized polypeptides are folded with the assistance of ATP-dependent cytosolic chaperones (Hsp70 and the barrel-shaped TRiC/CCT chaperonin). Misfolded or short-lived regulatory proteins are tagged with polyubiquitin chains by E1–E2–E3 ubiquitin ligases and degraded into short peptides by the cytosolic 26S proteasome.

How does the cell prevent cytosolic enzymes from aggregating in such a crowded (300 mg/mL) environment?

Cells deploy two mechanisms to prevent catastrophic protein aggregation in the crowded cytosol. First, globular proteins evolve negatively charged, hydrophilic surface residues that maintain repulsive electrostatic screening and a tightly bound hydration shell. Second, the cytosol maintains high concentrations of molecular chaperones (such as Hsp70 and Hsp90) and unexpectedly employs ATP at millimolar concentrations (2–8 mM) not only as an energy currency, but also as an amphiphilic biological hydrotrope that solubilizes hydrophobic protein patches and prevents pathological liquid-to-solid amyloid transitions.

Why does glycolysis occur in the cytosol while the citric acid cycle occurs inside mitochondria?

Glycolysis evolved prior to the Great Oxidation Event in the anaerobic Last Universal Common Ancestor (LUCA), making it native to the ancient prokaryotic cytosol. When the ancestral host archaeon engulfed the alphaproteobacterial endosymbiont that became the mitochondria, the aerobic citric acid cycle and oxidative phosphorylation remained inside the endosymbiont's matrix and inner membrane, while the host retained cytosolic glycolysis to feed pyruvate into the organelle.

What is cytoplasmic streaming (cyclosis) and why do large cells need it?

Brownian thermal diffusion is rapid over sub-micron bacterial distances (t ≈ x²/2D ≈ 0.5 ms across 1 μm), but scales with the square of distance—requiring hours to diffuse across a 1-millimeter plant cell or fungal hypha. Large eukaryotic cells overcome this diffusion bottleneck via cytoplasmic streaming (cyclosis), in which myosin motor proteins walk along cortical actin filament cables of the cytoskeleton, dragging organelles and stirring the bulk cytosol at velocities of 10 to 100 μm/s.