Microtubules are one of the principal components of the cytoskeleton that build up the structure and shape of the cell (Figure 1). They are also important in a number of cellular processes, such as cell division and intracellular transportation. Extending from a the centrosome, the major microtubule-organizing center (MTOC) in human cells, microtubules form a polar network extending toward the plasma membrane. This organization is highly conserved in evolution, reflected in a striking similarity of microtubules across almost all species (Janke C, 2014).
Of all human proteins, 430 proteins (2%) have been experimentally shown to localize to the microtubule cytoskeleton and its substructures; microtubule ends, cleavage furrow, cytokinetic bridge, midbody, midbody ring, and mitotic spindle (Figure 2 and 3). Functional enrichment analysis of the genes encoding microtubule-localizing proteins shows highly enriched GO-terms for biological processes related to cytoskeleton organization, cytoskeletal transport, cell division, and post-translational protein folding. More than half of the proteins detected at the microtubules also localize to other cellular compartments, most commonly to nucleoplasm, cytosol and vesicles.
Figure 1. Examples of proteins localized to the microtubules and its substructures. TUBA1A is the major component of the microtubules, here shown to localize to the microtubules in three different cell lines (detected in A-431, U-251 and U-2 OS cells). DTNBP1 is a component of the BLOC-1 protein complex required for biogenesis of lysosome-related organelles. This protein was previously not known to localize to microtubules. By using independent antibodies DTNBP1 is shown to localize to microtubules (detected in U2-OS cells). AURKB is a key regulator of mitosis by being part of the chromosomal passenger complex that ensures the correct orientation of the chromosomes during their segregation. AURKB is localized to the cytokinetic bridge (detected in U2-OS cells). CAMSAP2 is a microtubule minus end protein that is expected to be involved in the nucleation and polymerization of microtubules. This protein is localized to the microtubule ends (detected in U2-OS cells).
Figure 2. 2% of all human protein-coding genes encode proteins localized to microtubules. Each bar is clickable and gives a search result of proteins that belong to the selected category.
The structure of microtubules
Microtubules are physically robust polymers made up of α/β-Tubulin dimers assembled into 13 linear protofilaments that associate laterally around a hollow core. The tubules are about 25 nm in diameter and can grow very long. Microtubule filaments display a polar structure with one fast-growing plus-end of exposed β-subunits and one slow-growing minus-end of exposed α-subunits. Microtubules are highly dynamic structures due to their ability to polymerize or depolymerize rapidly from end-to-end, two counteracting processes that are both regulated by the binding of GTP. The ability of microtubules to maintain highly dynamic polymerization patterns is vital for the cell's ability to adapt its structural arrangements in response to different environmental conditions, and for mechanical processes. A characteristic feature of microtubules is that they never reach a steady-state length and thus remain constantly in the process of elongation (polymerization) or shrinkage (depolymerization), a phenomenon known as dynamic instability (Desai A et al, 1997; Conde C et al, 2009). In the Cell Atlas, several substructures of the microtubule cytoskeleton are classified; microtubule ends, cleavage furrow, cytokinetic bridge, midbody, midbody ring, and mitotic spindle (Figure 3).
Figure 3. Examples of the substructures of the microtubules. Midbody ring: APC2 is localized to the midbody ring (detected in U-2 OS cells). Cytokinetic bridge: KIF18A is a motorprotein of the kinesin family that regulates chromsosome aggregation and supresses centromere movements prior to anaphase, thus contributing to chromosome stability (detected in U-2 OS cells). Mitotic spindle: FAM83D is localized to the mitotic spindle (detected in A-431 cells).
The dynamics of microtubules are regulated by a group of microtubule-associated proteins (MAPs). In addition, microtubules are subjected to a number of different post-translational modifications that influence the structure in order to meet the requirements for their different functions, for example acetylation of lysine residues, detyrosination, glycylation and glutamylation (Janke C, 2014; Wloga D et al, 2010). A selection of proteins suitable to be used as markers for microtubules and the substructures are listed in Table 1.
Table 1. Selection of proteins suitable as markers for the microtubules structure or its substructures.
See the morphology of microtubules in human induced stem cells in the Allen Cell Explorer.
The function of microtubules
Similar to other cytoskeletal networks, a major function of the microtubule cytoskeleton is to supply mechanical strength to the cytoplasm and maintain the intracellular organization of organelles. Microtubules are also vital for cell migration and motility, as well as for intracellular transport of organelles, vesicles and proteins. Microtubules are also structural components of eukaryotic cilia and flagella, which can mediate cellular motility and extracellular transport of fluids. Indeed, there is a number of ATP-driven motor proteins that move along microtubules in order to actively transport proteins and vesicles, or in order to create forces and movements between microtubules, as seen in cilia and flagella. Dynein and kinesin are the two largest families of motor proteins, moving in direction towards the minus and the plus end of microtubules, respectively. The function in intracellular transportation makes the microtubule skeleton a key member of the secretory pathway, where it channels the post-Golgi vesicles out to the plasma membrane (Schmoranzer J et al, 2003).
Another highly important and well studied function of microtubules is in cell division through mitosis. Microtubules constitue a major part of the mitotic spindle (see Figure 3), which mediates segregation of sister chromatids to opposit poles. Spindle formation is an intricate process that involves both polymerization and depolymerization of microtubules, as well as movements generated by motor proteins. Sister chromatid separation is followed by cytokinesis, upon which microtubules of the central spindle are rearranged and compacted between the daughter cells, forming a cytokinetic bridge with a dense central structure called the midbody, which is eventually cleaved (see Figure 3) (Skop AR et al, 2004).
Several diseases are linked to defective cellular transport due to abnormalities in microtubules. Hereditary diseases associated with defects in cilia, known as ciliopathies, and several neurodegenerative disorders such as Parkinson's syndrome are two such diseases (Waters AM et al, 2011; Cappelletti G et al, 2015). Moreover, as tumour growth is highly dependent on mitosis, there are many efficient anti-cancer drugs that target microtubules (Jordan MA et al, 2004). Gene Ontology (GO)-based analysis of genes encoding proteins localizing to microtubules shows enrichment of functions highly in line with existing literature. The most highly enriched terms for the GO domain Biological Process are related to microtubule-based processes such as cytoskeleton organization, cilium morphogenesis and cell division (Figure 4a). Enrichment analysis of the GO domain Molecular Function also shows top hits for enriched terms related to motor activity and tubulin binding (Figure 4b).
Figure 4a. Gene Ontology-based enrichment analysis for the microtubules proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 4b. Gene Ontology-based enrichment analysis for the microtubules proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Table 2. Highly expressed microtubules proteins across different cell lines.
Microtubules proteins with multiple locations
Approximately 76% (n=327) of the microtubule-localizing proteins detected in the Cell Atlas also localize to other compartments in the cell (Figure 5). The network plot shows that the most common locations shared with microtubules are the nucleoplasm, the cytosol and vesicles. As microtubules are essential for intracellular transport, often utilized by molecules encapsulated in vesicles, the dual locations of these proteins reflect the important role of microtubules as a transport system in the cell.
Figure 5. Interactive network plot of microtubule proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to the microtubules and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the microtubule proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p<=0.05). Each node is clickable and results in a list of all proteins that are found in the connected organelles.
Expression levels of microtubules proteins in tissue
Transcriptome analysis and classification of genes into tissue distribution categories (Figure 6) shows that genes encoding microtubule-localizing proteins are less likely to be detected in all tissues, but more likely to be detected in many tissues, compared to all genes presented the Cell Atlas. This points towards a somewhat more restricted pattern and tissue expression of these genes.
Figure 6. Bar plot showing the percentage of genes in different tissue distribution categories microtubule-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Relevant links and publications
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.