Question

CELL BIOLOGY
Using microtubules as an example, explain the roles of tubulin dimer concentrations and bound GTP in the polymerization and depolymerization of microtubules in vitro. Within your answer, be sure to explain nucleation, critical concentrations, treadmilling, and catastrophe.

Answer 1

Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can grow as long as 50 micrometers and are highly dynamic. The outer diameter of a microtubule is between 23 and 27 nm while the inner diameter is between 11 and 15 nm. They are formed by the polymerization of a dimer of two globular proteins, alpha, and beta-tubulin into protofilaments that can then associate laterally to form a hollow tube, the microtubule. The most common form of a microtubule consists of 13 protofilaments in the tubular arrangement.

Microtubules are one of the cytoskeletal filament systems in eukaryotic cells. The microtubule cytoskeleton is involved in the transport of material within cells, carried out by motor proteins that move on the surface of the microtubule.

Microtubules are very important in a number of cellular processes. They are involved in maintaining the 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 macromolecular assemblies (see entries for dynein and kinesin. They are also involved in cell division (by mitosis and meiosis) and are the major constituents of mitotic spindles, which are used to pull eukaryotic chromosomes apart.

Microtubules are nucleated and organized by microtubule organizing centers (MTOCs), such as the centrosome found in the center of many animal cells or the basal bodies found in cilia and flagella, or the spindle pole bodies found in most fungi.

There are many proteins that bind to microtubules, including the motor proteins kinesin and dynein, microtubule-severing proteins like katanin, and other proteins important for regulating microtubule dynamics. Recently an actin-like protein has been found in a gram-positive bacterium Bacillus thuringiensis, which forms a microtubule-like structure called a nanotubule, involved in plasmid segregation. Other bacterial microtubules have a ring of five protofilaments.

The tubular structure of microtubules is sturdier than microfilaments, allowing a lot of heavy-duty pulling and pushing. Though sturdy, microtubules are temperature-dependent – they depolymerize if cooled to 4°C and will depolymerize again if heated to 37°C provided that GTP is available.

The basic building block of microtubules is an α-tubulin/β-tubulin dimer (encoded by TUBA_ genes and TUBB_ genes respectively). The individual tubulin proteins each weigh ~55kDa, so at ~110Da / amino acid that’s on the order of 500 amino acids. Both α- and β-tubulin bind GTP, but α pretty much just holds onto it forever, whereas in β it can be exchanged or hydrolyzed to GDP and then exchanged for new GTP again.

Strands of consecutive α/β dimers make up protofilaments, and 13 protofilaments arranged side-by-side into a cylinder make a microtubule. The space in between the protofilaments is called a ‘seam’. The whole microtubule is ~25 nm in diameter. You can kind of see its structure (made of dimers and protofilaments) in the Inner Life of the Cell segment about microtubules:

Nucleation

Nucleation is the event that initiates the formation of microtubules from the tubulin dimer. Microtubules are typically nucleated and organized by organelles called microtubule-organizing centers (MTOCs). Contained within the MTOC are another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits of the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a lock washer-like structure known as the "γ-tubulin ring complex" (γ-TuRC). This complex acts as a template 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.

The centrosome is the primary MTOC of most cell types. However, microtubules can be nucleated from other sites as well. For example, cilia and flagella have MTOCs at their base termed basal bodies. In addition, work from the Kaverina group at Vanderbilt, as well as others, suggests that the Golgi apparatus can serve as an important platform for the nucleation of microtubules. Because nucleation from the centrosome is inherently symmetrical, Golgi-associated microtubule nucleation may allow the cell to establish asymmetry in the microtubule network. In recent studies, the Vale group at UCSF identified the protein complex again as a critical factor for centrosome-dependent, spindle-based microtubule generation. It has been shown to interact with γ-TuRC and increase microtubule density around the mitotic spindle origin.

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

Treadmilling

Treadmilling is a phenomenon observed in many cellular cytoskeletal filaments, especially in actin filaments and microtubules. It occurs when one end of a filament grows in length while the other end shrinks resulting in a section of filament seemingly "moving" across a stratum or the cytosol. This is due to the constant removal of the protein subunits from these filaments at one end of the filament while protein subunits are constantly added at the other end. Two main theories exist on microtubule movement within the cell: dynamic instability and treadmilling. Dynamic instability occurs when the microtubule assembles and disassembles at one end only, while treadmilling occurs when one end polymerizes while the other end disassembles. However, the biological significance of treadmilling in vivo is not well characterized. This is due to the fact that within a living cell, many microtubules are tightly anchored at one end of the filament. Some research has suggested that the differences in critical concentration between the positive and the negative end may be a way for the cell to prevent unwanted polymerization events.

Critical concentration.

The critical concentration is the concentration of either G-actin (actin) or the alpha,beta-tubulin complex (microtubules) at which the end will remain in an equilibrium state with no net growth or shrinkage. What determines whether the ends grow or shrink is entirely dependent on the cytosolic concentration of available monomer subunits in the surrounding area.[8] Critical concentration differs from the positive (CC+) and the negative end (CC−), and under normal physiological conditions, the critical concentration is lower at the positive end than the negative end. Examples in how the cytosolic concentration relates to the critical concentration and polymerization are as follows:

• A cytosolic concentration of subunits above both the CC+ and CC− ends results in subunit addition at both ends
• A cytosolic concentration of subunits below both the CC+ and CC− ends results in subunit removal at both ends

Note that the cytosolic concentration of the monomer subunit between the CC+ and CC− ends is what is defined as treadmilling in which there is growth at the plus end, and shrinking on the minus end.

The cell attempts to maintain a subunit concentration between the dissociation constants at the plus and minus ends of the polymer.

catastrophe

A microtubule “catastrophe” event manifests itself by the sudden switch of a growing microtubule into a rapidly shortening state. The widely accepted view of microtubule catastrophe is that it involves a single random event, such as the sudden loss of a protective end structure.

A microtubule “catastrophe” event manifests itself by the sudden switch of a growing microtubule into a rapidly shortening state. The widely accepted view of microtubule catastrophe is that it involves a single random event, such as the sudden loss of a protective end structure . This single-step mechanism implies that a microtubule has the same probability of undergoing catastrophe at any given point in time, irrespective of how long it has been growing already. In this model, the `catastrophe frequency', which is the number of observed catastrophes divided by the total period of microtubule growth, remains constant over time, and the distribution of microtubule lifetimes and lengths is predicted to follow a decaying exponential distribution.