THE HUMAN CELL


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 other cellular processes, such as cell division and transportation. Extending from a central microtubule-organizing center, they display a polar structure that is highly conserved in evolution, reflected in a striking similarity of microtubules across almost all species (Janke C, 2014).

Of the proteins detected in the Cell Atlas, 372 proteins (2%) have been experimentally shown to localize to the microtubule cytoskeleton and its substructures; the cytokinetic bridge, microtubule ends, midbody, midbody ring and the mitotic spindle (Figure 2 and 3). Functional enrichment analysis of the microtubule-localizing proteins shows highly enriched GO-terms for biological processes related to cytoskeleton organization, cell movement and cell division (Figure 4). More than half of the proteins detected at the microtubules also localize to other cellular compartments, most commonly to nucleoplasm, cytosol and vesicles (Figure 5).

TUBA1A - A-431
TUBA1A - U-251 MG
TUBA1A - U-2 OS


DTNBP1 - U-2 OS
AURKB - U-2 OS
CAMSAP2 - U-2 OS

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).

  • 2% (372 proteins) of all human proteins have been experimentally detected in the microtubules by the Human Protein Atlas.
  • 86 proteins in the microtubules are supported by experimental evidence and out of these 20 proteins are validated by the Human Protein Atlas.
  • 262 proteins in the microtubules have multiple locations.
  • 110 proteins in the microtubules show a cell to cell variation. Of these 109 show a variation in intensity and 1 a spatial variation.
  • Proteins are mainly involved in cytoskeleton organization and cell division.

Figure 2. 2% of all human protein-coding genes encode proteins localized to the microtubules. Each bar is clickable and gives a search result of proteins that belong to the selected category.

The structure of the microtubules


Substructures

  • Microtubules: 265
  • Microtubule ends: 4
  • Cytokinetic bridge: 90
  • Midbody: 36
  • Midbody ring: 12
  • Mitotic spindle: 16

    Microtubules are physically robust polymers made up of α/β-Tubulin dimers called protofilaments assembled into groups of 13 around a hollow core about 25 nm in diameter. When these dimer complexes polymerize, they form linear microtubule filaments that display a polar structure with two growing ends. One fast-growing plus-end with exposed β-subunits and one slow-growing minus-end with exposed α-subunits. Microtubules display a highly dynamic structure 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 spatial arrangements in response to different environmental conditions, and to execute mechanical work. 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; the cytokinetic bridge, microtubule ends, midbody, midbody ring and the mitotic spindle (Figure 3).

    APC2 - U-2 OS
    CCSAP - U-2 OS
    C12orf66 - MCF7

    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: CCSAP is a spindle-associated protein that is localized to the cytokinetic bridge (detected in U-2 OS cells). Mitotic spindle: C12orf66 is localized to the mitotic spindle (detected in MCF-7 cells).

    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.

    Gene

    Description

    Substructure

    TUBA1A Tubulin, alpha 1a Microtubules
    DTNBP1 Dystrobrevin binding protein 1 Microtubules
    Midbody
    CAMSAP2 Calmodulin regulated spectrin-associated protein family, member 2 Cytosol
    Microtubule ends

    The function of the microtubules


    One major function of the microtubule cytoskeleton is to supply mechanical strength to the cytoplasm and maintain the intracellular organization of the organelles. Microtubules are vital for cell migration and for the mobility of organelles within the cell. They make up eukaryotic cilia and flagella, and act as substrates for motor proteins that can move along the filaments to convert chemical energy into mechanical energy, which facilitates movement. Dynein and kinesin are the two largest families of motor proteins. The dyneins move in direction towards the microtubule organizing center (MTOC) whereas the kinesins are directed away from the MTOC. As an important platform for organelle movement, the microtubule skeleton is a key member of the secretory pathway, where it channels the post-Golgi vesicles out to the plasma membrane. This is done by the help of motor proteins that attach to the vesicles (Schmoranzer J et al, 2003). Another highly important and well studied function of the microtubules is to participate in cell division. Here, they form the mitotic spindle (see Figure 3) in order to properly segregate the chromosomes during mitosis, a function with significant implications in cancer where the mitotic rate is out of control. During the last steps of mitosis, a protein structure called the midbody (see Figure 3) is compacted between the two daughter cells, held together by the cytokinetic bridge (see Figure 3) (Skop AR et al, 2004). Several diseases are linked to defective cellular transport due to abnormalities in the microtubules. Many efficient anti-cancer drugs therefore target the microtubules (Jordan MA et al, 2004). Hereditary diseases associated with defects in cilia, known as ciliopathies, and several neurodegenerative disorders such as Parkinson's syndrome are two of such diseases (Waters AM et al, 2011; Cappelletti G et al, 2015). Gene Ontology (GO)-based analysis of the corresponding genes in the microtubule proteome shows 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.

    Gene

    Description

    Average TPM

    TUBA1B Tubulin, alpha 1b 2341
    TUBB4B Tubulin, beta 4B class IVb 481
    TUBA1A Tubulin, alpha 1a 256
    TUBB Tubulin, beta class I 122
    BIRC5 Baculoviral IAP repeat containing 5 85
    TUBA4A Tubulin, alpha 4a 54
    TUBB2A Tubulin, beta 2A class IIa 53
    SPRY2 Sprouty RTK signaling antagonist 2 30
    VPS4A Vacuolar protein sorting 4 homolog A (S. cerevisiae) 26
    SKA1 Spindle and kinetochore associated complex subunit 1 13

    Microtubules proteins with multiple locations


    Approximately 70% (n=262) 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 the microtubules are essential for intracellular transport, often utilized by molecules encapsulated in vesicles, the dual locations of these proteins reflect the important role of the 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 (Figure 6) shows that microtubule-localizing proteins are less likely to be expressed in all tissues, compared to all genes with protein data in the Cell Atlas.

    Figure 6. Bar plot showing the distribution of expression categories, based on the gene expression in tissues, for microtubules-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks statistically significant deviation(s) (p≤0.05) from all other organelles 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


    Cappelletti G et al, 2015. Linking microtubules to Parkinson's disease: the case of parkin. Biochem Soc Trans.
    PubMed: 25849932 DOI: 10.1042/BST20150007

    Conde C et al, 2009. Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci.
    PubMed: 19377501 DOI: 10.1038/nrn2631

    Desai A et al, 1997. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol.
    PubMed: 9442869 DOI: 10.1146/annurev.cellbio.13.1.83

    Janke C. 2014. The tubulin code: molecular components, readout mechanisms, and functions. J Cell Biol.
    PubMed: 25135932 DOI: 10.1083/jcb.201406055

    Jordan MA et al, 2004. Microtubules as a target for anticancer drugs. Nat Rev Cancer.
    PubMed: 15057285 DOI: 10.1038/nrc1317

    Schmoranzer J et al, 2003. Role of microtubules in fusion of post-Golgi vesicles to the plasma membrane. Mol Biol Cell.
    PubMed: 12686609 DOI: 10.1091/mbc.E02-08-0500

    Skop AR et al, 2004. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms. Science.
    PubMed: 15166316 DOI: 10.1126/science.1097931

    Waters AM et al, 2011. Ciliopathies: an expanding disease spectrum. Pediatr Nephrol.
    PubMed: 21210154 DOI: 10.1007/s00467-010-1731-7

    Wloga D et al, 2010. Post-translational modifications of microtubules. J Cell Sci.
    PubMed: 20930140 DOI: 10.1242/jcs.063727