Microtubules are key components of the cytoskeleton and are composed of alpha and beta tubulin. These dimerise in a head to tail fashion to form 13 linear protofilaments. The protofilaments associate initially into sheets and subsequently into hollow tubes with a diameter of roughly 25 nm. These hollow tubes radiate from the microtubule-organising centre (MTOC) located at the centrosome in interphase cells.
Microtubules are essential in intracellular trafficking of vesicles and mitochondria, cell polarisation and migration. Furthermore, microtubules are involved in the development and maintenance of cell shape.
In mitosis, microtubules undergo dynamic instability, constantly growing and shrinking, to ensure accurate chromosome alignment and segregation. The mitotic spindle consists of polar microtubules that extend from opposite poles of the cell and interact with the chromosomes. These microtubules attach to specialized protein structures called kinetochores, located at the centromere of each chromosome, forming kinetochore microtubules. These interactions exert forces that align and separate the chromosomes during different stages of mitosis, such as metaphase and anaphase.
Moreover, microtubules also contribute to cytokinesis, the final stage of cell division. During cytokinesis, a contractile ring composed of actin and myosin filaments forms at the equator of the cell, constricting to divide the cell into two daughter cells. Microtubules help position and guide the placement of the contractile ring, ensuring proper division and distribution of cellular components.
Structure of microtubules
Microtubules and Mitosis
Microtubules are highly dynamic polymers that continuously grow and shrink during interphase and mitosis, with the rate of microtubule polymerisation to depolymerisation called the ‘catastrophe rate’. In many cell types, the microtubule network organises in a radial manner, with the minus end embedded close to nucleus and the positive end exploring the cytoplasm. To further regulate microtubule dynamics, various proteins are involved in the process. One such protein is the microtubule-associated protein (MAP), which binds to microtubules and influences their stability and organization. Different types of MAPs perform distinct functions, including promoting microtubule assembly or preventing excessive depolymerization. By interacting with microtubules, MAPs contribute to the overall integrity and regulation of the microtubule network.
During microtubule polymerisation GTP-loaded beta tubulin is added to the plus end of the microtubule. Shortly after the addition of GTP-loaded beta-tubulin to the plus end of the microtubule GTP is hydrolysed to GDP. Stabilisation of microtubules requires the maintenance of a GTP-bound beta-tubulin ‘cap’ to prevent depolymerisation. Removal of the ‘cap’ results in GDP-bound tubulin subunits adopting a curved conformation, the breaking of lateral bonds and microtubule catastrophe.
Furthermore, certain anti-cancer drugs target microtubules to disrupt their dynamics and inhibit cell division. Microtubule-targeting agents, such as taxanes and vinca alkaloids, interfere with microtubule polymerization or depolymerization, leading to mitotic arrest and subsequent cell death. These drugs have found applications in cancer chemotherapy and continue to be an active area of research in developing new therapies.
Understanding the intricate role of microtubules in mitosis not only provides insights into fundamental cellular processes but also holds implications for various pathological conditions, including cancer, neurodegenerative diseases, and developmental disorders. Continued research in this field aims to unravel the mechanisms governing microtubule dynamics and explore potential therapeutic interventions.
Mitosis (Cell Division Cycle)
Microtubule Organising Centres (MTOCs)
Microtubules are required for the accurate separation of chromosomes during mitosis and the maintenance of genetic integrity. During mitosis, microtubules form the mitotic spindle through the formation of a bipolar array of microtubules emanating from the centrosomes or the spindle pole body. Additionally, microtubules play a crucial role in the overall organization and structure of the mitotic spindle.
During prometaphase, microtubules emanate from the microtubule organizing centers (MTOCs) and extend and shorten until they become amphitelically attached to chromosomes at their kinetochores. This attachment is critical for ensuring proper alignment and distribution of chromosomes during subsequent stages of mitosis. Activation of the spindle assembly checkpoint (SAC) during prometaphase prevents cells from transitioning to anaphase until correct bipolar attachment of microtubules to chromosome kinetochores occurs.
However, if the correct microtubule-kinetochore attachment is not achieved in a timely manner due to monotelic, syntelic, or merotelic attachment of microtubules to kinetochores, the cells arrest in a pro-metaphase/metaphase-like state. This prolonged arrest provides an opportunity for error correction mechanisms to operate, ensuring accurate chromosome segregation. Failure to resolve defective microtubule-kinetochore attachment can eventually result in cell death or chromosomal instability.
Completion of metaphase and the onset of anaphase occur following satisfaction of the SAC, which is triggered by the correct attachment of microtubules to chromosomes. The SAC monitors the attachment status of each chromosome, ensuring that all chromosomes are properly aligned and attached before the cell progresses to anaphase. This coordination of events ensures the alignment of chromosomes along the metaphase plate and the subsequent separation of daughter chromosomes into two identical sets during anaphase.
This highly regulated process ultimately leads to the cleavage of the cell into two daughter cells, each receiving the correct genomic information. The fidelity of microtubule-mediated chromosome segregation is crucial for maintaining genetic stability and preventing chromosomal abnormalities that can lead to various diseases, including cancer.
Microtubule Targeting Agents (MTAs)
Microtubule dynamics during mitosis are a target of a diverse group of chemotherapeutics called the microtubule targeting agents (MTAs). MTAs have been used successfully over the past 30 years to treat a broad range of cancers including, ovarian, breast, lung and Kaposi’s sarcoma.
One group of MTAs, known as microtubule polymerizing agents, promotes microtubule polymerization by stabilizing their assembly. By binding to the microtubule ends, these agents enhance microtubule formation and prevent their disassembly, leading to mitotic arrest and subsequent cell death. Examples of microtubule polymerizing agents include taxanes such as paclitaxel and docetaxel, which have shown remarkable efficacy in treating various solid tumors. These drugs interfere with microtubule dynamics, ultimately inhibiting cancer cell proliferation and inducing apoptosis.
Conversely, the other group of MTAs, referred to as microtubule depolymerizing agents, disrupt microtubule formation by inducing their rapid disassembly. These agents bind to microtubules and promote their destabilization, resulting in the formation of abnormal mitotic spindles and ultimately leading to cell death. Vinca alkaloids like vincristine and vinblastine are well-known examples of microtubule depolymerizing agents used in cancer therapy. They exert their antitumor effects by interfering with microtubule dynamics, disrupting the mitotic process, and triggering apoptosis in cancer cells.
Taxol and Nocodazole
Taxol (Paclitaxel) was originally isolated from the bark of a yew tree (Taxus brevifola) by Monroe Wall and Mansukh Wani in 1967. It was approved for clinical use in 1995 and is now used to treat a range of malignancies such as breast cancer, kaposi’s sarcoma, ovarian cancer and nonsmall cell lung cancer. Taxol binds tubulin heterodimers with a 1:1 stoichiometry along the surface of microtubules, thus, stabilising microtubules and resulting in microtubule bundle formation.
The suppression of microtubule dynamics by MTAs results in the inhibition of microtubule spindle formation, kinetochore-microtubule attachment and prevents chromosome bi-orientation. This in turn leads to the activation of the SAC, prolonged mitotic arrest and subsequent cell death. However, to date the link between prolonged mitotic arrest and the initiation of cell death is poorly understood.
During mitosis chromosomes can attach Taxol-stabilised microtubules; however, due to the lack of microtubule dynamics, the correct tension is not established across sister chromatids, which prevents chromosome alignment and bi-orientation during metaphase. If complete chromosomal alignment is not achieved, cells undergo protracted mitotic arrest which eventually results in cell death . However, despite the widespread use of MTAs for a number of decades the molecular mechanism of action is still not completely understood. In particular the molecular components linking MTA-induced prolonged mitotic arrest to cell death has not been clearly defined.
Nocodazole, vincristine, and colchicine are a diverse group of agents that exhibit distinct structural characteristics and exert their effects on microtubule function through binding to different sites on β-tubulin. By interfering with microtubule dynamics or inducing microtubule depolymerization, these compounds have proven to be valuable tools in various research applications and cancer treatment.
One notable application of nocodazole is its frequent use in cell synchronization studies. Researchers utilize brief exposures to nocodazole to temporarily arrest cells in mitosis, allowing for the synchronization of cellular processes and facilitating the study of specific events during this phase of the cell cycle.
Vinca alkaloids, including vincristine, have shown efficacy in the treatment of various neoplasms, such as breast cancer. These compounds target microtubules and disrupt their normal function, impeding cancer cell growth and proliferation. By interfering with microtubule dynamics, vinca alkaloids exhibit potent antitumor effects and have become important components of chemotherapy regimens for several types of cancer.
The utilization of these agents in both research and clinical settings highlights the significant impact of understanding microtubule function and the potential for developing novel therapies targeting microtubule dynamics in the future. Ongoing research aims to further elucidate the mechanisms of action of these agents and explore their potential in combination with other treatments to improve patient outcomes in cancer therapy.
The Kinetochore Microtubule Network (KMN) platform
The KMN network is a supermolecular evolutionarily conserved basic unit of the kinetochore composed of four-subunits of the Ndc80 complex (Ndc80, Nuf2, Spc24, and Spc25), Mis12 complex (Dsn1, Nnf1, Nsl1, and Mis12) and the two-subunit KNL-1 complex (KNL-1 and Zwint). While mammals and yeasts have additional centromeric proteins (CCAN network), fly kinetochores are able to function simply with the KMN network of proteins. Recently the KMN network was constructed in vitro using a bacterial expression system, and was demonstrated to interact with centromeres through the Mis12 complex. The KMN network member SPC105 (KNL-1/ Blinkin/ CASC5/ DA40/ AF15q14) has been shown to recruit the mitotic checkpoint complex proteins BubR1, Bub1 and Bub3 which are required to restrain anaphase progression by inhibiting the spindle assembly checkpoint complex .
Another KMN network member Ndc80/Hec1 is required for the recruitment of the checkpoint proteins Zwint, Zw10 complex, and the Mad1-Mad2 complex to the kinetochore during mitosis, although it remains unclear if Mad1 interacts directly with Hec1. In addition to checkpoint proteins, both Ndc80 and SPC105 are also required for recruitment of microtubule associated proteins EB1, CLIP70 and p150Glued, thus suggesting that Blinkin and Ndc80 may act as a molecular bridge coupling the sensing of microtubule attachment to SAC signalling.
While spindle checkpoint proteins have been known for over a couple of decades, the precise biochemical nature of the signal that checkpoint proteins sense to distinguish a correct and incorrect attachment is unclear. Recent progress has identified that KMN network proteins interact with both checkpoint proteins and microtubules, thus setting the stage for addressing the complex question of how the KMN network processes microtubule attachment and signals to extinguish the SAC to allow for anaphase progression and cell division.
In conclusion, mitosis is a highly regulated process essential for cell division, and microtubules play a central role in orchestrating this complex event. The dynamic behavior of microtubules, including their polymerization, depolymerization, and organization into the mitotic spindle, is crucial for accurate chromosome segregation and the maintenance of genomic stability. The targeted disruption of microtubule dynamics has proven to be a successful strategy in cancer treatment, with microtubule targeting agents (MTAs) demonstrating efficacy in a wide range of cancers. Ongoing research aims to develop more selective and targeted MTAs, as well as exploring combination therapies, to enhance treatment efficacy and minimize adverse effects.
Written by Pragna Krishnapur
Pragna Krishnapur completed her Bachelor degree in Biotechnology Engineering in Visvesvaraya Technological University before completing her Masters in Biotechnology at University College Dublin.