The cytoskeleton sustains the cell shape, which could be polygon, fiber, oval or biconcave round. Without cytoskeleton, the cells must be spherical. The cytoskeleton also takes part in cell motility. Some cells, such as macrophage, hemamoeba and amoeba, can move by changing their shape. This style of movement is called amoeboid movements and dependent on cytoskeleton. The cytoskeleton also keeps the organelles in their position and plays a role in the transportation in a cell.

The cytoskeleton system is a three-dimensional network of protein fiber in eukaryotic cells. It distributes in cytoplasm and karyoplasm. There are three types of filaments in the cytoskeleton system: microtubule, microfilament and intermediate filament.

Microtubules (MTs) are the thickest in cytoskeleton system. Under electron microscope, microtubule looks hollow cylindrical. Microtubules have an external diameter of 24-26 nm, and an internal diameter of 15nm. Microtubules vary in length that could be several centimeters in motoneurons of central nervous system. Microtubules could exist as singlets, doublets or triplets.

A typical singlet microtubule consists of 13 protofilaments in a cylinder. The singlet microtubule is unstable microtubule and dynamically polymerizing and depolymerizing, which is the structural base for amoeboid movement and intercellular transportation of membrane vesicles and organelles. The Spindle fibers are singlet microtubules. They assemble during cell division and depolymerize thereafter. The microtubules in axons or dendrites of a neuron are parallel to the long axis of the neuron.

A doublet microtubule consists of an A tubule and a B tubule. The A tubule is complete singlet microtubule with 13 protofilaments, while the B tubule contains 10 protofilaments. The B tubule shares 3 protofilaments with the A tubule. The microtubules in the axoneme of cilium and flagellum are doublet microtubules. Doublet microtubules are stable microtubules.

A triplet microtubule contains one complete 13-protofilament A tubule fused to the incomplete 10-protofilament B tubule, which in turn is fused to the incomplete 10-protofilament C tubule. The B tubule shares 3 protofilaments with the A tubule, and the C tubule shares 3 protofilaments with the B tubule. Each triplet has 33 protofilaments. The centrosome and the microtubules in the fundus of cilium and flagellum are triplet microtubules. The triplet microtubules are stable microtubules.

The tubulin is a heterodimer of α and β subunits. In the heterodimer, there is a specific binding site for colchicine and vincristine, and a specific binding site for guanosine diphosphate (GDP) and guanosine triphosphate (GTP).

Microtubule-associated proteins (MAPs) are the auxiliary proteins that could bind to microtubules. Some MAPs can bind the ends of microtubules and regulate the stability and dynamics of microtubules. Some of the MAPs, such as MAP-1, MAP-2, Tau and MAP-4, link microtubules to other cell components. These MAPs have at least one basic microtubule-binding domain and one projection domain. The basic microtubule-binding domains can bind to the surface of microtubules and the projection domain may either bind to microtubules or other specific cell components. Tau proteins exist in the dendrites and axons of neuron. They cross-link the microtubules and keep them in organization. The axonemal dyneins bind the A tubule of a doublet microtubule with its stem part and the B tubule of another doublet microtubule. The head has ATPase activity and can hydrolyze ATP to supply energy for flagellum and cilium movement.

In a GTP-tubulin solution, the tubulin heterodimers would assemble to form microtubules and the microtubules would grow until the free tubulin heterodimers were consumed to the critical concentration. The assembly of microtubule could be divided to three phases.

Nucleation phase or lag phase. Heterodimers joint head-to-tail to form a protofilament. The heterodimers joint to the protofilament laterally to form sheets, and eventually form microtubules. The initiation of de novo formation of microtubules is called microtubule nucleation.

Polymerization phase or elongation phase. Both ends of a microtubule can either assemble or disassemble. But the assembly speed of the ends are different, showing the assembly polarity. In the microtubule, the tubulin dimers are arranged in the same direction with the β-tubulin side towards the plus (+) end. Typically, the plus end assembles faster than the minus (-) end.

Steady state phase or equilibrium phase. When the assembly and disassembly achieve a balance, the overall length of microtubules is relatively constant and growing and shrinking microtubules coexist. This is called dynamic instability. Some of the microtubules may grow up in one end and shrink down in the other end, which is known as treadmilling behavior.

Microtubule organizing center (MTOC), including the matrix, flagellum and flagellum, are the place that microtubules in cells start to assembly.

In MTOC, γ-tubulin ring complex (γ-TuRC) becomes the core of microtubule assembly. At the beginning, γ-TuRC forms a ring structure with 13 γ-tubulin molecules and the same diameter as the microtubule. And then heterodimers of α-tubulin and β-tubulin are added to this ring structure in a certain direction with the plus end of the microtubule outward. The γ-TuRC or other proteins may bind to and stabilizes the minus end of some microtubules. Some minus ends are free and may elongate or shorten.

Microtubule assembly could be affected to many factors, tubulin concentration, temperature, pH, divalent cation concentration, GTP concentration, MAPs and specific drugs.

Tubulin concentration. When the polymerization speed is equal to depolymerization speed, the tubulin concentration is called critical concentration, and microtubules display dynamic equilibrium. When the concentration is higher than critical concentration, the assembly will be faster. Otherwise, when the concentration is below critical concentration, the assembly will be slower.

Temperature and pH. Polymerization occurs when temperature is about 37℃ and depolymerization occurs when temperature is about 0 ℃. The optimum pH for microtubule assembly is 6.9.

Divalent Cation Concentration. Either decrease of Ca2+ concentration or increase of Mg2+ concentration can promote microtubule polymerization. Either increase of Ca2+ concentration or decrease of Mg2+ concentration can promote depolymerization.

GTP concentration. There are GTP binding sites in tubulin heterodimer. Each subunit of the heterodimer can bind a GTP molecule. The GTP bind to the α-tubulin is stable and considered as a part of the heterodimer. Heterodimers are activated when their β-subunits are bound with GTP. The GTP-tubulin has a straight conformation and higher affinity to the microtubule end. The GTP-tubulins can polymerize quickly and form tubulin-GTP cap in the microtubule end. Microtubules with tubulin-GTP cap grow rapidly. After a GTP-tubulin polymerizes into a microtubule, the GTP is hydrolyzed into GDP much faster, so the middle part of the microtubules are made up by GDP-tubulins. With the consumption of GTP-tubulins, polymerization speed of GTP-tubulins decreases and the tubulin-GTP cap shortens. Once a microtubule lost its tubulin-GTP cap, it began to shrink rapidly and the GDP-tubulins were released. The free GDP-tubulin has a curved conformation and could be transformed to GTP-tubulin, which could participate in polymerization again.

MAPs. Some MAPs can induce tubulin to polymerize and stabilize the microtubules. Some MAPs can regulate the movement of microtubules, and polymerization and depolymerization of tubulins.

Taxol and deuterium oxide (D2O). Taxol is extracted from the bark of yew tree. It is often used to treat breast cancer, ovarian cancer, lymphoma and multiple myeloma. Taxol and D2O can reduce the critical concentration and thus promote microtubule assembly and make microtubules concentrated in cluster. Taxol and D2O can bind to the polymerized heterodimers and stabilize the microtubules. As the microtubules are not able to disassemble, the spindle fibers will no longer tow the chromosomes and the cell division is blocked.

Colchicine. Colchicine is an alkaloid extracted from meadow saffron. It had already been used to treat gout in ancient Egypt. Colchicine is able to specifically bind to a tubulin heterodimer, change its conformation and interrupt its assembly. Colchicine can inhibit spindle formation and stop the mitosis.

Vincristine. Vincristine is an alkaloid extracted from Catharanthus roseus. It can be used as antineoplastic and has good curative effect on acute lymphatic leukemia. Vincristine, similar to colchicine, can bind tubulin heterodimer and restrain microtubule polymerization. Vincristine has greater effect and different binding site compared to colchicine.

Microtubules take part in the supporting network of cells, maintain the shape of cells and fix the position of organelles. The biconvex discoid structure of platelet is maintained by the ring-shaped vascular bundles that array around the platelet. The forming and maintaining of axon and dendrite depends on microtubules. Some microtubules are closely linked with other organelles. Some ribosomes are attached on the intersection points of microtubules and microfilaments. Mitochondria distribute around the long axis of microtubule.

Centrioles are rod-shaped corpuscles that mainly composed of tubulin. A pair of orthogonally arranged centrioles and the surrounding pericentriolar material (PCM) makes up a centrosome. The centrosome is the main microtubule organizing center of animal cells. The microtubule arrays extending from the centrosome form a star-like structure named aster.

Each centriole is composed of 9 triplet microtubules. The triplet microtubules array obliquely, which looks like the wings of a windmill from the transverse section.

Cilia and flagella are special structures that extend out of the cells. Flagella are longer but less than cilia. Flagella are commonly seen on animal sperms. Cilia are often seen on some kinds of epithelia cells. By swinging, the cilia on the surface of respiratory tract can expel sputum and those on the surface of uterine tube can move oocytes. Cilia and flagella have two parts, axoneme and basal body.

The axoneme of motile cilium or flagellum consists of 9 doublet microtubules and two singlet microtubules in the central sheath. The dynein binds tightly to an A microtubule with its tail and binds the B microtubule of nearby doublets with its heads. There is radial spokes between the central sheath and the out doublets. The stalk of radical spoke is linked to the A microtubule. The big end of the radical spoke is its head. The two central singlet microtubules are linked by inner bridges like rungs on a ladder. The axoneme of rotary and immobile cilium has no central singlets or radical spokes. The axoneme of immobile primary cilium lacks dynein, central singlets and radical spokes. The microtubules near the tip of the axoneme become singlets and integrated with each other. The basal body consists of 9 triplet microtubules. The triplets array obliquely and look like the wings of a windmill.

The ATPase activity of the dynein head hydrolyze ATP to supply energy for the movement of a cilium or flagellum. When moving, the B microtubules slide relatively to the A microtubules, the cilium or flagellum bends or swings regularly. The cilia on respiratory tract epithelia and esophagus epithelia can swing regularly in a special direction and make a liquid stream in the tubule, which expel the secretions, solid particles or impurity.

Primary ciliary dyskinesia (PCD) is a genetic disorder that causes defects in the motility of cilia in the respiratory tract and fallopian tube, as well as in the flagella of sperms. The cilia or flagella of the patients lack dyneins. The patients suffer from infertile because their sperms cannot move. Meanwhile, they are susceptible to chronic recurrent respiratory infections, as the ability of mucus clearance is damaged.

Microtubules supply tracts for transportation of organelles and vesicles in a cell. The motor protein families, kinesins and dyneins, are the proteins that mediate the transportation. The head of the motor integrates with the microtubule and the tail of the motor binds the receptor linked with the vesicle or organelle. The head of motor protein has ATPase activity. After hydrolyzing ATP, the head changes its conformation and finishes the binding/release cycle, which makes the organelle or vesicle moving along the microtubule.

In interphase, the duplicated centrioles remain together near the nucleus. When the cell enters M phase, these two pairs of centrioles split and move apart to form the two poles of the mitotic spindle. The spindle pole serves as the main microtubule organizing center (MTOC) for the spindle microtubules, known as spindle fibers. The plus end of the spindle fiber projects away from the spindle pole, while the minus end is fixed on the spindle pole. The inherent dynamic instability of the plus ends of the spindle fibers allows them to search the cellular space for chromosomes or other binding sites. The chromosome can stabilize and organize microtubules. Some of the microtubules may find the kinetochore, which is a specialized protein structure on the surface of the centromere, and get attached and stabilized. The capture of microtubules by the kinetochore is facilitated by the chromatin-mediated microtubule nucleation. The kinetochore-mediated and microtubule-mediated microtubule nucleation contributes to the growth of mitotic spindle as well. In the metaphase, the kinetochore microtubules disassemble in the minus ends and assemble in the plus ends at similar speed, which keep the length of the kinetochore microtubules constant roughly. After the chromatids are separated in the anaphase, the kinetochore microtubules disassemble in the plus ends and the kinetochore moves along the microtubules. So the force for the chromosome movement is a combination of the microtubule shortening and the translocation of the microtubules.

The axoplasmic flow tract is composed by microtubules. In the cerebral cortex neuron of the patients suffering from Alzheimer's disease (AD), there has accumulated a lot of distorted and twisted microtubules and tau proteins. The tau protein is hyperphosphorylated and its affinity to microtubule declines, which lowers the microtubule stability, causes deficiency to the gathering of microtubules, and eventually results in insoluble neurofibrillary tangles, blocking of the axoplasmic flow and thus interrupting the signaling.

Microfilament (MF) is the thinnest solid fiber in the cytoskeleton. Microfilaments are composed of double-stranded helix fiber and 5-8 nm in diameter. They can be either cross-linked to form networks or arranged in parallel to form bundles.

The actin subunit, also called globular actin or G-actin, is folded into two domains that unit each other with an ATP or ADP molecule between them. In human, there are three isoforms of actin, termed α, β, and γ. α-Actin is expressed in muscle cells only, while β- and γ-actin are expressed together in almost all non-muscle cells. In fibroblasts cultured in vitro, all three isoforms of actins are present. Eukaryotic actins are extremely conserved with more than 90% identity between any pair of actins, which indicates that they are evolved from a common ancestor. The G-actins assemble head-to-tail to form a filament, which is called filamentous or F-actin.

Actin-binding proteins (ABPs) regulate the assembly of actins. Monomer sequestering proteins bind to G-actin, inhibit G-actin polymerization and keep a pool of G-actins. Crosslinking proteins organize actin fibers into networks or bundles. Filament-capping proteins bind to one end or both ends of an actin filament and inhibits either growing or shortening of actin filaments. Filament-Severing/Depolymerization Proteins sever actin filaments, control the length of F-actins, and reduce the viscosity of the cell. Membrane Binding Proteins connect actin fibers to cell membrane and serve for stretching and contracting in non-muscle cells.

The assembly of microfilament could be divided to three phases, nucleation phase, growth phase, and equilibrium phase.

Nucleation Phase. At first, three G-actins aggregate to be the core. Then, G-actin monomers are rapidly added to the two ends of the trimer, which extends the fiber into F-actin. Two F-actins are twisted into helix, i.e., microfilament (MF). Because all actin monomers point in the same direction, the filaments are polar.

Growth Phase. While both ends of actin filaments can add new G-actins, the growing speeds could vary, up to 10 times, which displays assembly polarity. The plus (+) end, also referred to as barbed end, extends faster than the minus (-) end, which is also called pointed end.

Equilibrium Phase. Once the concentration of G-actins declines to the critical concentration, the polymerization rate equals the depolymerization rate, actin filaments stay in the same length. When the G-actins undergo a net assembly at the plus end and a net disassembly at the minus end, the filaments undergo treadmilling.

Microfilament assembly could be affected by many factors, G-actin concentration, divalent cations, pH, ATP, actin binding proteins, and specific drugs.

When G-actin concentration is above the critical concentration, the polymerization happens. When G-actin concentration is below the critical concentration, the filaments shorten. When the G-actin concentration is equal to the critical concentration, the overall length of the filaments will stay unchanged.

Either Mg2+ or high concentrated K+ promotes assembly; Either Ca2+ or low concentrated K+ promotes depolymerization.

pH higher than 7.0 promotes polymerization. When the pH is too low, actin monomers will never assemble.

The actin could bind either an ATP or GDP molecule. ATP-actin has a lower critical concentration at the plus end than that of the minus end. The critical concentrations for ADP-actin are the same in both ends and higher than that of ATP-actin. During the polymerization process, ATP that binds to G-action is hydrolyzed in two steps: cleavage of ATP followed by slower release of Pi. The ADP. Pi-F-action are stable, but the ADP-F-action are not stable. There will be an ADP. Pi-actin cap at the rapidly polymerizing end of the microfilament. With the releasing of Pi, some filaments may lost the ADP. Pi-actin cap and shorten quickly. Some filaments may grow in one or both ends. But the overall length of the filaments keep consistent at the steady sate. This phenomenon is called dynamic instability.

Actin assembly is regulated by actin-binding proteins, some of which fix actin filaments like a nail, and some of which bundle the actin filaments like a rope.

Cytochalasin B is an alkaloid produced by mold. It can bind to the end of microfilaments, prevent the addition of new G-actin and thus inhibit assembly.

Phalloidin, a highly toxic alkaloid extracted from the mushroom (poisonous mushrooms), has a strong affinity to actin filaments. It can stabilize actin and inhibit depolymerization.

Microfilaments usually support small projections of cells, for example, microvilli of intestinal epithelial cells. The microfilament bundles, crosslinked by fimbrin and villin, in the axis center of microvillus maintain the shape of the microvillus. The base of the microvillus is deeply embedded in the terminal web of intercrossing actin filaments.

Stress fibers are formed by actin bundles, non-muscle myosin II, and accessory proteins. They play important roles in cell adhesion, migration and morphogenesis.

Hereditary spherocytosis (HS), an autosomal dominant (AD) genetic disorder, is caused by mutations in genes coding the proteins that make the shape of red blood cells. The membrane of normal red blood cell is composed of actin, spectrin, ankyrin, band 3 proteins, glycophorin, and many other contractible proteins that interconnected to form a film of skeleton network. The skeleton network maintains the biconcave disc shape of erythrocyte, which enlarge the surface of the red blood cell and strengthen its deformation capacity. As a results of that the gene mutations cause defects of membrane skeleton, the membrane skeleton lose contractile function, the red blood cells become spherical, and the deformability of red blood cells declines. The red blood cells became fragile and prone to rupture, which results in hemolytic anemia.

Amoeboid Movement. The front end of the cell extends forward the pseudopod, the cytoplasm follows, and then the rear end withdraws gradually, which moves the whole cell forward. The polymerization and depolymerization of microfilaments contribution to the filopodia formation and deformation. The white blood cells will move to sites of inflammation from the blood vessel by amoeboid movement.

Membrane Ruffling. Membrane ruffling could be observed in monolayer cells cultured in vitro. In the bottom surface, the cell membrane ruffles, folds and protrudes alternately, which is due to the contraction of the microfilaments under the membrane.

Cyclosis, i.e., cytoplasmic streaming, is caused by the moving of myosin-coated organelles along microfilaments, which makes the material flow along a certain direction, and keeps the even distribution of various nutrients and metabolites within the cell.

Some endocytosis and exocytosis processes of cell are related to the contraction of the microfilament mesh under cell membrane. During some endocytosis, the film under the wire contracts, cell depresses, cell membrane melts and breaks, and the substances are ingested. During exocytosis, microfilaments shrink under the membrane, gap appears in micro screen, secretory vesicles get close to and integrated with the plasma membrane, and the substances are emitted.

During telophase, bundles of actin filaments form a contractile ring confined near the equator. The contractile ring constantly depolymerizes, shrinks, and becomes smaller and smaller, until eventually complete the cytokinesis.

Involved in Muscle Contraction

The contractile unit of a skeletal muscle cell, myofibril, is composed of two types of myofilaments, the thick filaments and the thin filaments. The thick filaments consists mainly of myosin II, while the thin filament consists of actin and regulatory proteins tropomyosin and troponin.

The cross-section of skeletal muscle shows that each thick filament is surrounded by six thin filaments. When muscle contracts, the lengths of thick and thin filament are constant, thick filaments and thin filaments slide over one another.

During the fertilization, the sperm cell membrane contacts with the glial layer of the egg surface, which triggers the acrosome reaction: the membrane of acrosome fuses with the membrane of the sperm, the hydrolytic enzymes were released to degrade the surface of the egg glial layer, the acrosome grows rapidly to form the acrosome spike, which gets contact with the oocyte membrane and causes the fusion of sperm and egg cells. The formation of acrosome spike is related to rapid actin polymerization.

Microfilament network may serve as a scaffold for many signal transduction systems and hold them at or near the inner face of cell membrane.

In the process of the embryonic development of advanced creatures, some cells are constantly migrating and reshaping. For example, the neural plate is reshaped to neural groove and then neural tube. The cells in the top circle of this part usually have a parallel actin ring respectively. When the microfilaments contract, the upper cells become small and wrinkled while the lower cells do not change, which makes the plate curling up.

The Intermediate filament (IF) is first so called, in muscle, because that its diameter, about 10nm, is intermediate between those of the thin and thick filaments of striated muscle. This name is generally accepted for all IFs as the diameter is also between those of microfilaments and microtubules. Some of the IFs distribute under the nucleus membrane and interweave into meticulous fiber lattice.

IF is made up of IF proteins. An IF protein molecule consists of a head, a stem and a tail. The stem is a highly conserved coiling region. This region consists of ~310-350 amino acids. The head, the amino terminal, and the tail, the carboxyl terminal, are non-helical and may vary dramatically in both amino acid sequence and length, which contributes to the diversity of IFs. IF proteins are classified into 6 types based on the sequence homology: Keratin, Vimentin, Desmin, Neurofilament, Glial Fibrillary Acidic Protein and Lamins. The identity of the amino acid sequences of IF proteins in the same homology group is up to 70-90%.

Keratins are heterodimers of an acidic (Type I) and a basic (Type II) intermediate filament polypeptide. Keratins distribute in simple and glandular epithelia. It is divided into two types, α and β. Alpha keratins form tenacious structures, such as hair and nail. Beta keratins are distributed in cell plasma of epithelial tissues, such as keratinized epidermis, and epithelium of esophagus, airway and bile duct. In epithelial cells, keratin intermediate filaments connect with desmosomes.

Vimentin, belonging to Type III, distributes in mesenchymal cells and cells originating from mesenchymal cells, such as blood cells and lymphocytes. However, vimentin is expressed in all kinds of cells cultured in vitro. Vimentin looks dispersal and curly. Vimentin network extend from the nuclear envelope to the cell membrane, in some cases connected to desmosome and hemidesmosome. Vimentin play roles in conferring mechanical resistance to cells, organelle positioning, cell migration and adhesion, and cell signaling.

Desmin, belonging to Type III, distributes in muscle. Desmin intermediate filaments connect muscle cells to each other.

Neurofilaments, belonging to type IV, distribute in the neurons of central and peripheral nervous systems, both in cell body and axon. In the axon, neurofilaments always align with microtubules to form a complex, which makes the nerve fiber to be elastic.

Glial fibrillary acidic protein (GFAP), belonging to type III, is expressed in glial cells of central nervous system and sustains the shape of the cells.

Lamins, belonging to type V, form meshwork underneath the nuclear membrane and distribute in the nucleoplasm. Lamin intermediate filaments take part in breakdown and reassembly of nuclear envelope.

Intermediate Filament Associated Proteins (IFAPs) cross-link intermediate filaments closely or loosely and mediate the fasciculation or reticulation of IFs.

Individual intermediate filament proteins are aligned in parallel and wound together with their rod domains to form heterodimer or homodimer. Two of these dimers assemble, in an antiparallel and staggered manner to form a tetramer. The tetramer is apolar. Eight tetramers associate together to form the unit-length filament (ULF). The ULFs anneal longitudinally to form the intermediate filaments. The mature IFs are flexible and could be stretched to more than three folds of their lengths.

IF Assembly has several characteristics. IFs are stable, do not disassemble into monomers, and do not have treadmilling phenomenon. IFs are apolar and can grow in both ends. No specific drug has been found to affect the assembly yet. In vitro, self-assembly of IFs does not depend on ATP or GTP.

IFs keep the location of nucleus, against the gravitational descending, as the IFs abound around the nucleus and extend to cytoplasm and extracellular matrix. IFs exist throughout the nucleus and cytoplasm, forming an integral supporting system.

At the site of cell junction, such as tight junction, desmosome and hemidesmosome, there are IFs involved in to enhance the linkage of cells, tolerate shearing force and mechanical stress.

Epidermolysis bullosa simplex (EBS), a disease caused by keratin gene mutations that destroy the keratin intermediate filament network, causes the skin to be very fragile and blister easily. The sufferer is sensitive to mechanical injury. Even a slight crushing can damage the basal cells and cause vesication of the skin.

The lamina intermediate filaments take part in the breakdown and reassembly of nuclear envelop during cell division.

Neurofilament, distributed in dendrites and axons of nerve cell, plays a cradle role in transportation of metabolites in nerve cell.

The expression of IFs is tissue specific. Different types of cell express different IFs, which could be used to distinguish epithelium, muscle, mesenchyme, glial cell and neuron.

The tissue specific expression of IFs is maintained in tumorigenesis and even metastasis. Thus, the antibodies of IFs could be used to identify the origin of tumor cells, which is very important to identify the primary cancer and treatment of cancer.

For example, the feature of epithelial tumor is keratin, the feature of myogenic tumor is desmin, the feature of mesenchymal tumor is vimentin, the feature of neural tumor is GFAP, and the feature of neuroblastoma is neurofilament protein.

During amniocentesis, if the infant had malformation of central nervous system, there should be cells with GFAP or neurofilaments in amniotic fluid, doctor can determine it by identification of IF proteins.

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