17 Microtubule poisons
17.1 Introduction to the cytoskeleton
We will now discuss drugs that target microtubules (MTs). There are two types of drugs represented by vinca alkaloids such as vincristine, and the taxanes, represented by paclitaxel. To understand how MT targeting drugswork, we will start this section by explaining what MTs are. We will briefly talk about the many cellular functions MTs play and will finally explain how do MT inhibitors work. MTs are a component of the cytoskelton. The cytoskeleton provides the cell with structure and shape. There are three types of filaments in the cytoskeleton such as actin filaments, intermediate filaments, and MTs.
Figure 15.1 shows animal cells in interphase, which are stained with fluorescent labels to help visualise the cytoskeleton. MTs are coloured in green, actin filaments are shown in red, and the nucleus containing DNA is shaded in blue. Actin filaments act for example as tracks for myosin motors and generate contractile forces both in muscle as well as non-muscle cell types. Intermediate filaments organise the three-dimensional structure of the cells, for example anchoring organelles. And finally MTs, which have many important cellular functions. In this image you can observe the prominent and extended cellular network MTs form. We will concentrate on MT filaments.
17.2 Microtubules: multifunctional protein assembly with key cellular roles
MTs have many essential cellular functions. Here I can only mention a few important ones. This is by no means a comprehensive description of the many cellular functions of MTs.
- As mentioned in the previous slide, they are involved in maintaining the structure of cells and the cell shape.
- They also provide the platform for intracellular transport. For example, members of two motor protein families, the dyneins and kinesins, move along MTs in either the plus or minus direction to unidirectionally transport a variety of cargos (Figure 15.2).
- Another very important function is the involvement of MTs in meiosis and mitosis, for example in the formation of the bipolar spindle or chromosome alignment and segregation.
17.3 Microtubules structure and dynamics
MTs are highly dynamic structures composed of α,β-tubulin subunits (Figure 15.3). β-tubulin is shown in purple and α-tubulin in blue. They form stable heterodimers. The heterodimers assemble into linear protofilaments. A single MT is comprised of 10–15 protofilaments, usually 13 in mammalian cells that associate laterally to form a 24 nm wide hollow cylinder. In each protofilament, the heterodimers are oriented with their β-tubulin monomer pointing towards the faster growing plus end and their α-tubulin monomer exposed at the slower growing minus end. MT growth is initiated by γ-tubulin and capping proteins. MTs can either grow by the addition of tubulin heterodimers, driven by GTP hydrolysis, a process called polymerisation. Or MTs can shrink or depolymerise as shown on the right. This polymerisation and depolymerisation behaviour is responsible for the highly dynamic nature of MTs in the cell. MTs are particularly dynamic during mitosis, during which they turn over in about 20 min.
17.4 Tubulin/microtubules as key players during mitosis: an excursion
We have learned that cancer chemotherapy targets dividing cells. Before looking at MT-targeting agents, we have to recapitulate, how cells divide. Therefore, we will have to briefly look at the cell cycle, the motor of cell proliferation.
We have already talked about the cell cycle in a previous chapter (see chapter 6) and we will briefly add a few important remarks. The cell cycle is a series of events that take place in cells leading to duplication into two daughter cells (Figure 15.4). The cell cycle is divided into two main stages: interphase and mitosis. Interphase is further subdivided into the G1 phase, S phase (or Synthesis) and the G2 phase.
- During G1, the cell grows and the duplication of organelles occurs.
- During S phase, DNA replication occurs and chromosomes are duplicated.
- During G2, cells continue to grow and prepare for mitosis.
- During mitosis, the cell finally divides. We will go into more detail of the mitotic phase in a moment.
Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. An important function of checkpoints is to assess DNA damage. In case DNA damage is detected the cell either stalls the cell cycle to repair the damage or, if repairs cannot be made, targets the cell for destruction via the apoptotic pathway. This is important for the mechanism of action of alkylating agents and Cisplatin, as we have heard.
- During G1, the G1 checkpoint ensures that everything is prepared for DNA synthesis.
- The G2 checkpoint verifies that everything is ready to enter mitosis.
- The mitotic checkpoint makes sure that all chromosomes are properly aligned at the metaphase plate.
It is also important to realise that different drugs may target different phases of the cell cycle.
MTs are essential for cell division, in particular mitosis and cytokinesis (Figure 15.5). Centrosomes and MTs are coloured in yellow and chromosomes are shown in blue.
- During prophase, the duplicated centrosomes start to separate on the surface of the nuclear envelope.
- In prometaphase, the bipolar spindle starts to form and chromosomes are captured by kinetochore MTs.
- In metaphase, centrosomes are at the two spindle poles and chromosomes are aligned at the metaphase plate.
- During anaphase, the sister chromatides start to separate, by moving to the opposite spindle poles.
- During telophase and cytokinesis, the genetic information is equally distributed between the two daughter cells, which will physically separate at the end of the process.
How can you remember the different stages of mitosis? Memorise the following sentence: I Prommised Marriage to Anna by Telephone.*