Chapter 4: Cytoskeleton



The cytoskeleton is a complex network of protein filaments that establish a supportive scaffolding system within the cell (Figure 4.1). Cytoskeletal proteins are located throughout the interior of the cell, anchored to the plasma membrane, the outer boundary of the cell, and traversing the cytoplasm (the interior contents of the cell bound by the plasma membrane). Organelles reside within the framework established by the cytoskeleton.

The cytoskeleton is not simply a passive internal skeleton but is a dynamic regulatory feature of the cell. Microtubules are one type of cytoskeletal protein. They organize the cytoplasm and interact with organelles to induce their movement. In addition to microtubules, actin filaments and intermediate filaments constitute the cytoskeleton. These components of the cytoskeleton work together as an integrated network of support within the cytoplasm.

FIGURE 4.1.The cytoskeleton as intracellular scaffolding.

The cytoskeleton as intracellular scaffolding.

Each type of cytoskeletal filament is formed from a specific association of protein monomer subunits. Actin filaments and microtubules are formed from globular protein subunits (more compact), while intermediate filaments contain fibrous protein subunits (more extended). Accessory proteins regulate the filament length, position, and association with organelles and the plasma membrane.


First isolated from skeletal muscle, actin was originally believed to be a protein found exclusively in muscle tissues. Some forms of actin (four of the six known forms) are found only in muscle cells, while other forms of actin are found within the cytoplasm of most cell types. Actin is now also believed to be present within the nucleus of most cells. Actin has a diameter of approximately 8 nm and forms structures known as microfilaments. Along with microtubules, actin helps to establish a cytoplasmic protein framework that can be visualized radiating out from the nucleus to the lipid bilayer of the plasma membrane (Figure 4.2). Actin is often localized to regions near the plasma membrane referred to as the cell cortex.

FIGURE 4.2.Localization of cytoskeletal components.

Localization of cytoskeletal components.

Actin is important in inducing contraction in muscle cells. Functions of actin in the cytoplasm of nonmuscle cells include regulation of the physical state of the cytosol (the liquid portion of the cytoplasm without organelles), cell movement, and formation of contractile rings in cell division. Changes in the structure of actin from globular subunits to elongated polymerized microfilament structures regulate such functions in the cell. Within the nucleus, actin is important for chromatin and nuclear structure and is believed to be involved in the regulation of gene transcription.


Actin microfilaments in the cytoplasm are filamentous or F-actin structures that are polymers formed from individual monomers of G (globular) actin. The polymerization of G-actin subunits into an F-actin molecule is an energy-dependent process.

1.  Overview: Energy in the form of adenosine triphosphate (ATP) is needed for the addition of each G-actin monomer onto a growing F-actin molecule. Each individual G-actin monomer added to a growing F-actin molecule has an ATP molecule bound to it. After the G-actin monomer has polymerized onto the F-actin polymer, ATP is hydrolyzed to adenosine diphosphate (ADP) with the release of inorganic phosphate (Pi) (Figure 4.3). The ADP remains bound to individual G-actin monomers within F-actin polymers. An F-actin filament is composed of two strands of identical G-actin monomers with a structure reminiscent of two strands of beads wound around each other in a regular pattern (Figure 4.4). The ends of F-actin filaments are nonidentical. The plus (+) end is where the G-actin monomers bound by ATP add to a growing filament and the minus (-) end is where the subtraction of ADP-bound G-actin monomers occurs in a shrinking filament.

FIGURE 4.3.ATP hydrolysis in actin polymerization.

ATP hydrolysis in actin polymerization.

 FIGURE 4.4.Structure of F-actin.

Structure of F-actin.

2. Steps: The F-actin microfilament structure is generated in three phases: (i) lag, (ii) polymerization, and (iii) steady state (Figure 4.5). For the lag phase, three G-actin monomers with bound ATP join together to form a nucleation site onto which the growing F-actin filament is built. During the polymerization phase, new G-actin monomers are added to the growing chain, adding preferentially to the + end. ATP is hydrolyzed to ADP + Pi. A steady state is reached when addition of G-actin monomers to the + end occurs at the same rate as other G-actin monomers are removed from the – end, and the length of the F-actin polymer is maintained. The G-actin monomers that leave an F-actin polymer rejoin the cytoplasmic pool of unpolymerized G-actins. The process of addition and subtraction of G-actin monomers is described as “treadmilling.” If one observes a certain G-actin monomer during the polymerization process, it will appear to be moving along the length of the F-actin polymer (Figure 4.6). As additional G-actin monomers add onto the polymer, they seem to push the G-actin. Over time, the G-actin monomer of interest appears to have moved farther and farther along the F-actin polymer before falling off the other end.

FIGURE 4.5.Polymerization of actin.

Polymerization of actin.

FIGURE 4.6.Treadmilling of F-actin.

Treadmilling of F-actin.

Fungal products and actin

Some products produced by fungi are known to influence actin polymerization. Phalloidin is the toxin from the poisonous mushrooms Amanita phalloides. These mushrooms are often called “death caps” or “angels of death” owing to the toxic effects on persons who consume them. Early symptoms are gastrointestinal in nature and are followed by a latency period during which the individual feels better. However, between the fourth and eighth day after the consumption of a mushroom of this type, the person will experience liver and kidney failure and death. Early intervention and detoxification are sometimes helpful but liver transplantation may still be needed. Phalloidin functions by disrupting the normal function of actin.

It binds to F-actin polymers much more tightly than to G-actin monomers and promotes excessive polymerization while preventing filament depolymerization. It also inhibits the hydrolysis of ATP bound to G-actin monomers that have polymerized onto an F-actin polymer. Phalloidin is known to inhibit cell movement due to its inhibition of actin. Phalloidin can be used in the laboratory as an imaging tool to identify actin (see Figure 4.2). The cytochalasins are other fungal products that are useful laboratory tools. Because they are known to block polymerization of actin and cause changes in cell morphology, they can be used to inhibit cell movement and cell division and to induce programmed cell death.

Actin-binding proteins

Actin-binding proteins regulate the structure of actin within the cell, controlling the polymerization of G-actin monomers, bundling of microfilaments, and breakdown into smaller fragments as needed by the cell. Some actin-binding proteins interact with individual G-actin monomers and prevent their polymerization onto an F-actin polymer. Others bind to the assembled microfilaments and cause them either to bundle or cross-link with other microfilament chains or to fragment and disassemble. The complexity of actin-based structures within the cytoplasm regulates certain cell characteristics.

1. Regulators of the gel/sol of the cytosol: One characteristic of a cell is the physical nature of its cytosol. It can be described either as gel, a more firm state, or sol, a more soluble state. The more structured the actin, the firmer (gel) the cytosol. The less structured (more fragmented) the actin, the more soluble (sol) the cytosol. Actin is continuously treadmilling in both the gel and sol states, contributing to the character of the cytoplasm. In addition, actin-binding proteins regulate the structures of actin and therefore the state of the cytosol (Figure 4.7). A pool of G-actin monomers is in equilibrium with F-actin polymers and also with G-actin monomers bound to a sequestering protein that inhibits the ability of G-actin monomers to polymerize. F-actin can be broken into smaller sized fragments by the twisting action of the protein cofilin that also prevents further lengthening. F-actin polymers can also be made into more complex structures by the actions of bundling and cross-linking proteins. Those complex structures can be fragmented when needed, by actin-severing proteins such as gelsolin, resulting in a more sol state of the cytosol. Calcium is required for the function of actin-binding proteins that fragment actin polymers, and higher levels of calcium are found in the cytosol where the sol state is developing. For example, the cytosol of a phagocytic cell, such as a macrophage, may need to become less structured with more solubilized cortical actin in order to engulf an invading organism. And, a migrating cell such as a fibroblast moving along a substrate must polymerize the actin at the leading end to pull the cell forward. At the same time, it must solubilize its cortical actin to allow for the flow of contents behind the leading end (Figure 4.8).

FIGURE 4.7.Actin and the gel-sol transition.

Actin and the gel-sol transition.

FIGURE 4.8.Actin polymerization and cell movement.

Actin polymerization and cell movement.

2. Spectrin: Stability along with strength and support is imparted to cells by actin-binding proteins of the spectrin family. Particularly important in erythrocytes (red blood cells), spectrin has a long, flexible rod-like shape and exists in dimers. On the cytosolic side of the plasma membrane, spectrin dimers bind to F-actin filaments in an ATP-dependent manner to strengthen and support the erythrocyte membrane. A lattice-like arrangement of actin and spectrin exists with other proteins including ankyrin and protein 4.1 facilitating their interaction. The spectrin-actin association is important in maintaining the characteristic biconcave shape of erythrocytes that is important for maximizing the hemoglobin and oxygen carried by each red blood cell (Figure 4.9) (see LIR Biochemistry, Chapter 3). Spectrin deficiency results in a condition known as hereditary spherocytosis (Figure 4.10) with spherical erythrocytes that are fragile and susceptible to lysis. Erythrocytes of affected individuals lack the central pallor characteristic of normal, biconcave erythrocytes. Instead, their erythrocytes are spherical and are susceptible to lysis, resulting in hemolytic anemia.

FIGURE 4.9.The spectrin membrane skeleton in an erythrocyte.

The spectrin membrane skeleton in an erythrocyte.

FIGURE 4.10.Spectrin and erythrocyte shape.

Spectrin and erythrocyte shape.

Dystrophin: Alterations in other actin-binding proteins, such as dystrophin, can also result in disease. In skeletal muscle cells, dystrophin and related proteins form the dystrophin-glycoprotein complex that links actin to the basal lamina (see Chapter 2). This association between dystrophin and actin provides tensile strength to muscle fibers and also appears to act as a framework for signaling molecules. Defects in dystrophin result in muscular dystrophy (MD), a group of genetic disorders whose major symptom is muscle wasting

Contractile functions in nonmuscle cells

While actin polymerization helps to propel a cell forward (see Figure 4.8), contraction is needed to pull the plasma membrane of the lagging end away from the substrate to enable forward progress. In fact, contraction or tightening and shortening to produce a pulling force is needed to maintain cell structure and to carry out normal cellular functions. Actin participates in such contractions owing to the effects of an ATP-hydrolyzing motor protein of the myosin family.

1. Myosin: As is true for actin, myosin was first discovered in muscle but is found in all cell types. Actin-myosin interactions in muscle are well studied (see LIR Physiology). It is believed that similar mechanisms function to produce contractions in nonmuscle cells. Myosin molecules have a head domain that interacts with F-actin and a tail containing an ATP-binding site. They hydrolyze ATP when they bind to actin. Myosin’s interactions with actin are cyclic. Myosin binds to actin, detaches, and then binds again. The myosin tail can also attach to cellular structures and pull them along the actin filament. Several forms of myosin exist. In nonmuscle cells, myosin II is important for many examples of contraction as it slides actin filaments over each other to mediate local contractions (Figure 4.11A). Myosins I and V are involved in moving cellular cargo along the tracks provided by F-actin.

FIGURE 4.11. Contractile functions of actin in nonmuscle cells.

Contractile functions of actin in nonmuscle cells.

(A) Myosin II slides actin filaments over each other to mediate local contractions.(B) A contractile ring pinches a dividing cell into two daughter cells.

Forms of muscular dystrophy

When the dystrophin protein is absent or is present in a nonfunctional form, degeneration of muscle tissue results. Muscle wasting then follows when the ability to regenerate the muscle is exhausted. Duchenne MD (DMD) and Becker MD are both caused by mutations in the dystrophin (dys) gene, a large (2.6 Mbp) gene with 97 exons. Additionally, both are inherited as X-linked recessive traits, with male individuals expressing the disease when their only X chromosome carries the mutated dys. These forms of MD differ in age of onset and in severity. Symptoms of DMD are noticeable in early childhood and quickly become debilitating. Becker MD is characterized by slowly progressive muscle weakness in the pelvis and legs.

It has a later age of onset than DMD and less severe symptoms. Several large deletions in the dys gene are found in individuals with Becker MD. These deletions, however, do not result in a frameshift (see Chapter 9). Instead, internal portions of the gene are missing but transcription and translation of the remainder of the gene occur. A partially functional dystrophin protein is therefore synthesized, causing less severe muscle wasting. In contrast, in DMD, several smaller deletions are found in the dys gene. In DMD, however, there is a frameshift, resulting in early termination of the protein (see Chapter 9). No functional dystrophin is produced and more severe muscle degeneration occurs.

2. Structural and functional needs for contraction: Providing stability to cell structure is an important reason for actin-myosin–based contractions to occur. Myosin II interacts with F-actin in the cell cortex to stiffen it and help prevent deformation of the plasma membrane. In addition, actin bundles that encircle the inner portion of epithelial cells form a tension cable, known as a circumferential belt that can regulate cell shape. This type of contraction is important in wound-healing because the gap of the wound can be sealed via contraction of the existing cells. Cell division in all cell types also depends on contraction. Actin-myosin structures are important during cytokinesis, or the cytoplasmic division following nuclear division of mitosis (see Chapter 20). Here, an actin-myosin–based structure, called a contractile ring, is formed. The diameter of the contractile ring progressively decreases, deepening the cleavage furrow in order to pinch the cell into two daughter cells. The ATP hydrolysis by myosin attached to actin causes pulling on the actin and pinching and separation of the membrane (Figure 4.11B).