Microtubules

Microtubules

Microtubules are a component of the cytoskeleton, found throughout the cytoplasm. These tubular polymers of tubulin can grow as long as 50 micrometres, with an average length of 25 µm[1] and are highly dynamic. The outer diameter of a microtubule is about 25 nm while the inner diameter is about 12 nm. They are found in eukaryotic cells and are formed by the polymerization of a dimer of two globular proteins, alpha and beta tubulin.

Microtubules are very important in a number of cellular processes. They are involved in maintaining structure of the cell and together with microfilaments and intermediate filaments, they form the cytoskeleton. They also make up the internal structure of cilia and flagella.They provide platforms for intracellular transport and are involved in a variety of cellular processes, including the movement of secretory vesicles, organelles, and intracellular substances (see entries for dynein and kinesin).[2] They are also involved in cell division (mitosis and meiosis) including the formation of mitotic spindles, which are used to pull apart eukaryotic chromosomes.

Microtubules are nucleated and organized in microtubule organizing centres (MTOCs), such as the centrosome or the basal bodies found in cilia and flagella. These MTOCs may or may not possess centrioles.

There are many proteins that bind to microtubules, including motor proteins such as kinesin and dynein, severing proteins like katanin, and other proteins important for regulating microtubule dynamics.[3]


Structure

Microtubules are long, hollow cylinders made up of polymerised α- and β-tubulin dimers.[4]

The tubulin dimers polymerize end to end in protofilaments, which are the building block for the microtubule structure. Thirteen protofilaments associate laterally to form a single microtubule and this structure can then extend by the addition of more protofilaments. In order for polymerization to occur, dimers must be present at a concentration above a specific minimum called the critical concentration, although the process is accelerated by the addition of nuclei, which are lengthened.

Microtubules have a distinct polarity which is important for their biological function. Tubulin polymerizes end to end, with the α-subunits of one tubulin dimer contacting the β-subunits of the next. Therefore, in a protofilament, one end will have the α-subunits exposed while the other end will have the β-subunits exposed. These ends are designated the (−) and (+) ends, respectively. The protofilaments bundle parallel to one another, so, in a microtubule, there is one end, the (+) end, with only β-subunits exposed, while the other end, the (−) end, has only α-subunits exposed. Elongation of microtubules typically only occurs from the (+) end.[5]

The lateral association of the protofilaments generates an imperfect helix with one turn of the helix containing 13 tubulin dimers, each from a different protofilament. The image at the top of this article illustrates a small section of a microtubule, a few αβ dimers in length. The number of protofilaments can vary; microtubules made up of 14 protofilaments have been seen in vitro.[6]

Organization within cells

Microtubules are part of a structural network (the cytoskeleton) within the cell's cytoplasm. The primary role of the microtubule cytoskeleton is mechanical support, although microtubules also take part in many other processes. A microtubule is capable of growing and shrinking in order to generate force, and there are motor proteins that allow organelles and other cellular components to be carried along a microtubule. This combination of roles makes microtubules important for organizing cell layout.

Microtubule nucleation

Microtubules are typically nucleated and organized by dedicated organelles called microtubule-organizing centres (MTOCs). MTOCs associated with the base of a eukaryotic cilium or flagellum are typically termed basal bodies; otherwise MTOC's are called centrioles. In many cell types microtubules are primarily nucleated at MTOCs; however, there are many exceptions to this rule.

Cilia and flagella

Microtubules have a major structural role in eukaryotic cilia and flagella. Cilia and flagella always extend directly from a MTOC, in this case termed the basal body. The action of motor proteins on the various microtubule strands which run along a cilia or flagellum allow the organelle to bend and generate force for swimming, moving extracellular material, and other roles.

Note that prokaryotes do not possess tubulin or microtubules. Prokaryotic (both bacterial and archaeal) flagella are entirely different in structure from eukaryotic flagella.

Organization during cell division


Mitotic spindle

A notable structure involving microtubules is the mitotic spindle, used by most eukaryotic cells to segregate their chromosomes correctly during cell division. The mitotic spindle includes the spindle microtubules, associated proteins, and the MTOC. The microtubules originate in the MTOC and fan out into the cell; each cell has two MTOC's, as shown in the diagram.

The process of mitosis is facilitated by three main subgroups of microtubules, known as astral, polar, and kinetochore microtubules. An astral microtubule is a microtubule originating from the MTOC that does not connect to a chromosome. Astral microtubules instead interact with the cytoskeleton near the cell membrane and function in concert with specialized dynein motors. Dynein motors pull the MTOC toward the cell membrane, thus assisting in correct positioning and orientation of the entire apparatus.

Kinetochore microtubules directly connect to the chromosomes, at the kinetochores. To clarify the terminology, each chromosome has two chromatids, and each chromatid has a kinetochore; the two kinetophores are linked. The complex created by the two kinetochores on a chromosome is called the centromere.

The polar microtubules from one MTOC intertwine with the microtubules from the other MTOC; motor proteins make them push against each other and assist in the separation of the two daughter cells.

Midbody

Cell division in a typical eukaryote finishes with the generation of a final cytoplasmic bridge between the two daughter cells termed the midbody. This structure is built of microtubules that originally made up part of the mitotic spindle.

Nucleation and growth

Microtubules are often nucleated at a dedicated microtubule-organizing centre. Contained within the MTOC is another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits, which compose the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a circular structure known as the "γ-tubulin ring complex" (γ-TuRC). This complex acts as a scaffold for α/β-tubulin dimers to begin polymerization; it acts as a cap of the (−) end while microtubule growth continues away from the MTOC in the (+) direction.[7]

Cells lacking MTOCs

Some cell types, such as plant cells, do not contain MTOCs. In these cells, microtubules are nucleated from discrete sites in the cytoplasm. Other cell types, such as trypanosomatid parasites, have a MTOC but it is permanently found at the base of a flagellum. Nucleation of microtubules for structural roles and for generation of the mitotic spindle is not from a canonical centriole-like MTOC. The regulation of the microtubule cytoskeleton in these cells is an intense area of study.

Dynamic instability

Dynamic instability refers to the coexistence of assembly and disassembly at the (+) end of a microtubule. The microtubule can dynamically switch between growing and shrinking phases in this region.[8] During polymerization, both the α- and β-subunits of the tubulin dimer are bound to a molecule of GTP.[4] The GTP bound to α-tubulin is stable and it plays a structural function in this bound state. However, the GTP bound to β-tubulin may be hydrolyzed to GDP shortly after assembly resulting in the addition of new dimers. The kinetics of GDP-tubulin are different from those of GTP-tubulin as GDP-tubulin is prone to depolymerization.[9] A GDP-bound tubulin subunit at the tip of a microtubule will fall off, although a GDP-bound tubulin in the middle of a microtubule cannot spontaneously pop out. Since tubulin adds onto the end of the microtubule only in the GTP-bound state, there is a cap of GTP-bound tubulin at the tip of the microtubule, protecting it from disassembly. When hydrolysis catches up to the tip of the microtubule, it begins a rapid depolymerization and shrinkage. This switch from growth to shrinking is called a catastrophe. GTP-bound tubulin can begin adding to the tip of the microtubule again, providing a new cap and protecting the microtubule from shrinking. This is referred to as "rescue".[10]

The search and capture model of microtubule function

Microtubule plus ends are often localized to particular structures. As mentioned previously, they are found at kinetochores and used to pull chromosomes apart during mitosis. In polarized interphase cells, microtubules are disproportionately oriented from the MTOC towards the site of polarity, such as the leading edge of migrating fibroblasts. This configuration is thought to help deliver microtubule-bound vesicles from the Golgi to the site of polarity. In 1986 Marc Kirschner and Tim Mitchison proposed that microtubules used their dynamic properties of growth and shrinkage at their plus ends to probe the three dimensional space of the cell. Plus ends that encounter kinetochores or sites of polarity become captured and no longer display growth or shrinkage. In contrast to normal dynamic microtubules, which have a half-life of 5–10 minutes, the captured microtubules can last for hours. This idea is commonly known as "the search and capture" model.[11] Indeed, work since then has largely validated this idea. At the kinetochore, a variety of complexes have been shown to capture microtubule (+)-ends.[12] Moreover, a (+)-end capping activity for interphase microtubules has also been described.[13] This later activity is mediated by formins,[14] the adenomatous polyposis coli protein and EB1,[15] a protein that tracks along the growing plus ends of microtubules.

Summary of tubulin’s polymerization properties

The process of adding or removing monomers depends on the concentration of αβ-tubulin dimers in solution in relation to the critical concentration (Cc), which is the equilibrium constant for the dissociation of the dimers at the end of the microtubule.

  • If their concentration is greater than the Cc the microtubule will polymerize and grow. If the concentration is less than Cc then the length of the microtubule will decrease.
  • The Cc will vary depending on whether a cap of GTP or GDP is present, which in turn means that the (+) end will have a different value from the (-) end. In the same way that actin filaments grow, the (+) end is defined as the end where preferential growth occurs.
  • The dynamic activity at the (+) end is greater as it has a lower specific Cc.
  • With αβ-tubulin levels greater than the Cc the dimers will mainly accumulate at the (+) end.
  • When the αβ-tubulin concentration is greater than the Cc of the (+) end but below that of the (-) end growth will only occur in one direction, with subunits being added to one end and subunits dissociating from the other end.

The microtubule can therefore grow at both ends or only at one, depending on the concentrations of αβ-tubulin dimers. The interaction of the (-) end with MTOC will greatly decrease its activity.

These characteristics are derived from the existence of the microtubule’s dynamic instability, which means that in the same cell some microtubules are depolymerizing (catastrophe) and others are polymerizing (recovery).

Microtubule associated proteins

“In vivo” microtubule dynamics vary considerably. Assembly, disassembly, and catastrophe rates depend on which microtubule-associated proteins (MAPs) are present. Classical MAPs are classified by their molecular weight into two groups:

  • MAPs with a molecular weight below 55-62 kDa

They are also called τ(tau) proteins. They line the microtubule and form links with adjacent microtubules.

  • MAPs with a molecular weight of 200-1000 kDa

There are four known types of the heavier molecular weight MAPs: MAP-1, MAP-2, MAP-3 and MAP-4. MAP-1 proteins are a set of three different proteins: A, B and C. The C protein plays an important role in the retrograde transport of vesicles and is known as cytoplasmic dynein.

MAP-2 proteins are located in the dendrites and in the body of neurons, where they bind with other filaments.

The MAP-4 proteins are found in the majority of cells and they stabilize the microtubules.

Besides the classic MAPs there are other MAP types:

  • Novel MAPs that bind the length of the microtubules, just like classic maps. Examples include STOP (also known as MAP6), and enscosin (also known as MAP7).

Chemical effects on microtubule dynamics

A great number of drugs are able to bind to tubulin and modify its activation state. This will have the effect of interfering with microtubule dynamics. These drugs can have an effect at intracellular concentrations much lower than that of tubulin. This interference with microtubule dynamics can have the effect of stopping a cell’s cell cycle and can lead to programmed cell death or apoptosis. The compounds that modify tubulin’s activity can be divided into two general groups: polymerization inhibitors, such as colchicine and depolymerization inhibitors.

The drugs that can alter microtubule dynamics include:

  • The cancer-fighting taxane class of drugs (paclitaxel, [taxol] and docetaxel) block dynamic instability by stabilizing GDP-bound tubulin in the microtubule. Thus, even when hydrolysis of GTP reaches the tip of the microtubule, there is no depolymerization and the microtubule does not shrink back.
  • Eribulin binds to the (+) growing end of the microtubules. Eribulin exerts its anticancer effects by triggering apoptosis of cancer cells following prolonged and irreversible mitotic blockade.

Environmental effects on microtubule integrity

Microtubule polymers are extremely sensitive to various environmental effects. Very low levels of free calcium can destabilize microtubules and this prevented early researchers from studying the polymer in vitro.[4] Cold temperatures can also cause rapid depolymerization of microtubules.

Post-translational modification of microtubules

Although most microtubules have a half-life of 5-10 min, certain microtubules can remain stable for hours.[13] These stabilized microtubules accumulate post-translational modifications on their tubulin subunits by the action of microtubule-bound enzymes.[16][17] However, once the microtubule depolymerizes, most of these modifications are rapidly reversed by soluble enzymes. Since most modification reactions are slow while their reverse reactions are rapid, modified tubulin is only detected on long-lived stable microtubules. Most of these modifications occur on the C-terminal region of alpha-tubulin. This region, which is rich in negatively charged glutamate, forms relative unstructured tails that stick out of the microtubule and form contacts with motors. Thus it is believed that tubulin modifications regulate the interaction of motors with the microtubule. Since these stable modified microtubules are typically oriented towards the site of cell polarity in interphase cells, this subset of modified microtubules provide a specialized route that helps deliver vesicles to these polarized zones. These modifications include:

  • Detyrosination: the removal of the C-terminal tyrosine from alpha-tubulin. This reaction exposes a glutamate at the new C-terminus. As a result, microtubules that accumulate this modification are often referred to as Glu-microtubules. Although the tubulin carboxypeptidase has yet to be identified, the tubulin—tyrosine ligase (TTL) is known.[18]
  • Delta2: the removal of the last two residues from the C-terminus of alpha-tubulin.[19] Unlike detyrosination, this reaction is thought to be irreversible and has only been documented in neurons.
  • Acetylation: the addition of an acetyl group to lysine 40 of alpha-tubulin. Interestingly this modification occurs on a lysine that is only accessible from the inside of the microtubule. It remains unclear how enzymes access the lysine residue. The nature of the tubulin acetylase remains controversial; however the reverse reaction is known to be catalyzed by HDAC6.[20]
  • Polyglutamylation: the addition of a glutamate polymer (typically 4-6 residues long[21]) to the gamma-carboxyl group of anyone of five glutamtes found near the end of alpha-tubulin. Enzymes related to TTL add the initial branching glutamate (TTL4,5 and 7), while other enzymes that belong to the same family lengthen the polyglutamate chain (TTL6,11 and 13).[17]
  • Polyglycylation: the addition of a glycine polymer (2-10 residues long) to the gamma-carboxyl group of anyone of five glutamtes found near the end of beta-tubulin. TTL3 and 8 add the initial branching glycine, while TTL10 lengthens the polyglycine chain.[17]

Tubulin is also known to be phosphorylated, ubiquitinated, sumoylated and palmitoylated.[16]

Motor proteins

In addition to movement generated by the dynamic instability of the microtubule itself, the fibres are substrates along which motor proteins can move. Some proteins take advantage of the hydrolysis of ATP in order to generate mechanical energy and move substances along the microtubules. The major microtubule motor proteins are kinesin, which moves toward the (+) end of the microtubule, and dynein, which moves toward the (−) end.

  • Dynein has a similar structure to kinesin: it is composed of two identical heavy chains, which make up two globular heads, and a variable number of intermediate and light chains. Dynein mediated transport takes place from the (+) end towards the (-) end of the intra-microtubular canal. It has been suggested that ATP hydrolysis takes place in the globular heads. Dynein transports vesicles and organelles and in order to do this it has to interact with their membranes, for which it needs a protein complex, which contains a number of elements including dynactin.


  • The majority of the kinesins are involved in the transport of vesicles from a microtubule’s (-) end towards the (+) end, that is towards the distal part of a cell or neurite.

Intracellular viral transport

Some viruses (including retroviruses, herpesviruses, parvoviruses, and adenoviruses) that require access to the nucleus to replicate their genomes attach to the motor proteins (dynein), which transport them at 1-4 μm/s to the centrosome, near the nucleus.

Other functions

Microtubules play a part in biological processes in addition to their structural role as a component of the cytoskeleton (along with actin and the intermediate filaments).

In development

The cytoskeleton formed by microtubules is essential to the morphogenetic process of an organism’s development. For example, a network of whole, polarized microtubules is required to be present within the oocyte of Drosophila melanogaster during its embryogenesis in order to establish the axis of the egg. Signals sent between the follicular cells and the oocyte (such as factors similar to epidermal growth factor) cause the reorganization of the microtubules so that their (-) ends are located in the lower part of the oocyte, polarizing the structure and leading to the appearance of an anterior-posterior axis.[22] This involvement in the body’s architecture is also seen in mammals.[23]

Another area where microtubules are essential is the formation of the nervous system in higher vertebrates, where tubulin’s dynamics and those of the associated proteins (such as the MAPs) is finely controlled during the development of the brain's neuronal base.[24]

Regulation of gene expression

The cellular cytoskeleton is a dynamic element that functions on many different levels: in addition to giving it a particular form and supporting the transport of vesicles and organelles it can also influence gene expression. However, the signal transduction mechanisms involved in this communication are little understood. Notwithstanding this, the relationship between the drug-mediated depolymerization of microtubules and the specific expression of transcription factors has been described, which has provided information on the differential expression of the genes depending on the presence of these factors.[25] This communication between the cytoskeleton and the regulation of the cellular response is also related to the generation of growth factors: for example, this relation exists for connective tissue growth factor.[26]

This fact has a vital inconsistency in cancer treatments as paclitaxel (sold under the trademark taxol, a widely used antineoplastic drug) acts on cytoskeletal microtubules and it is their interaction with elements that regulate the cell cycle that provokes, in the presence of antineoplastic drugs, a series of cellular failures in the cancerous cells that lead to planned cell death or apoptosis.[27]

Additional images

References

External links

  • MBInfo - Formation and Function of Microtubules
  • 3D microtubule structures in the EM Data Bank(EMDB)