3.1 Microtubule Organization in Different Phases of the cell cycle
3.2 Linkage between nuclear transport and microtubule assembly in fission yeast
3.3 Mechanisms of microtubule reorganization in cultured human cells
In eukaryotic cells, microtubules are organized in the cytoplasm to form a network array during interphase (non-mitotic phase). The network array is rapidly reorganized into the mitotic spindle when cells enter mitosis (M phase) (Fig. 3-1). The network array of microtubules in interphase serves as trafficking trails for intracellular transport, while the spindle plays an essential role for chromosome segregation. Thus, microtubules play essential roles by changing their organization according to the stage of the cell cycle. Although textbooks describe many facts about the structure and functions of microtubules, it remains largely unknown regarding the molecular mechanisms by which cytoplasmic microtubules are reorganized into the spindle.
Figure 3-1 Organization of microtubules differs in interphase and in mitosis (M phase) of the cell cycle
During interphase, a network array of microtubules is formed in the cytoplasm. Upon entry into mitosis, the cytoplasmic array disappears and is reorganized into the spindle. We aim to elucidate the molecular mechanisms of the reorganization.
We are investigating the molecular mechanism of microtubule reorganization using fission yeast as a model system. In yeast, the nuclear envelope persists during mitosis (which is called “closed mitosis”), and the spindle is assembled in the nucleus (Fig. 3-2). The time and location of microtubule assembly are strictly controlled: microtubules are formed in the cytoplasm during interphase, while in the nucleus during mitosis. We therefore hypothesized that the temporal reorganization of cytoplasmic microtubules into the spindle is coordinated with the nucleocytoplasmic transport system.
Many researches represented by biochemical experiments using eggs of Xenopus laevis (frog) identified that the small GTPase Ran, which had been known as a master regulator of nucleocytoplasmic transport system, also promotes microtubule assembly.
Ran exists in a GDP-bound form (Ran-GDP) in the cytoplasm, whereas in a GTP-bound form (Ran-GTP) in the nucleus (Fig. 3-2). In the nuclear transport machinery, Ran-GTP is responsible for the unloading of cargo proteins in the nucleus. In higher organisms, however, the nuclear envelope breaks down upon entry into mitosis, and therefore the nucleocytoplasmic transport is no longer necessary (Fig. 3-2). Although Ran does not have to play any roles in mitosis, why does Ran have a different role, which is to promote microtubule assembly?
Figure 3-2 Interplay between nucleocytoplasmic transport and microtubule organization
In interphase, GTP-bound Ran (Ran-GTP) is abundant in the nucleus, while GDP-bound Ran (Ran-GDP) is in the cytoplasm. Ran regulates nucleocytoplasmic transport. In cells of higher organisms, the nuclear envelope breaks down upon entry into mitosis, but Ran-GTP plays another function: assembly of microtubules. In yeast, the nuclear envelope persists even during mitosis. therefore, the nuclear transport occurs even during mitosis.
Then we go back to the story of yeast. Since the nuclear envelope persists during mitosis in yeast, we hypothesized that Ran promotes spindle assembly by accumulating factors that induce microtubule formation in the nucleus (Fig. 3-2). Thus, yeast Ran plays an essential role even during mitosis to accommodate spindle assembly factors in the nucleus. We envisage that the role of Ran in promoting spindle assembly evolved from yeast, and the function of Ran remained even after evolution of having the nuclear envelope break down.
To induce spindle assembly, what kind of target proteins would Ran need to accumulate in the nucleus in fission yeast? We found that the microtubule-associated protein Alp7, the ortholog of human TACC (Transforming Acidic Coiled-Coil) protein accumulates in the nucleus only during mitosis, and experimentally characterized Alp7 as a target protein of Ran that promotes microtubule formation in the nucleus during mitosis (Fig. 3-3).
Related Publication:
Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast
Masamitsu Sato and Takashi Toda
Nature (2007) 447(7142):334-7. DOI:10.1038/nature05773
Interdependency of fission yeast Alp14/TOG and coiled-coil protein Alp7 in microtubule localization and bipolar spindle formation
Masamitsu Sato, Leah Vardy, Miguel Angel Garcia, Nirada Koonrugsa and Takashi Toda
Molecular Biology of the Cell (2004) 15:1609-22. DOI:10.1091/mbc.e03-11-0837
Deletion of Mia1/Alp7 activates Mad2-dependent spindle assembly checkpoint in fission yeast
Masamitsu Sato, Nirada Koonrugsa, Leah Vardy, Sylvie Tournier, Jonathan B. Millar and Takashi Toda
Nature Cell Biology (2003) 5:764-766. DOI:10.1038/ncb0903-764
Is spindle formation in fission yeast specific to the spiecies?: from the viewpoint of nuclear transport and spindle pole body
Masamitsu Sato, Mika Toya and Takashi Toda
PROTEIN, NUCLEIC ACID AND ENZYME (2008) 53(3):197-206.
※article only in Japanese
Why does Alp7 accumulate in the nucleus specifically during mitosis? We found that the cyclin-dependent kinase CDK1/Cdc2 phosphorylates Alp7 upon mitotic entry, which enhances the interaction between Alp7 and Importin α (nuclear transport factor). The nuclear transport of Alp7 is thus accelerated, which results in promotion of spindle assembly (Fig. 3-3).
Figure 3-3 Alp7 is accumulated in the nucleus during mitosis through phosphorylation by CDK1
(Left) In interphase, the microtubule-associated protein Alp7 shuttles between the nucleus and the cytoplasm. Even when Alp7 is imported in the nucleus, it is immediately exported to the cytoplasm, and therefore Alp7 predominantly localizes to the cytoplasm. As a result, microtubule assembly is promoted in the cytoplasm. (Right) Upon entry into mitosis, Alp7 is accumulated in the nucleus through phosphorylation by the cyclin-dependent kinase CDK1. The translocation of Alp7 is crucial for microtubule reorganization upon mitotic entry.
Related Publication:
Spatiotemporal regulation of nuclear transport machinery and microtubule organization
Naoyuki Okada and Masamitsu Sato
Cells (2015) 4(3):406-426. DOI:10.3390/cells4030406
CDK-dependent phosphorylation of Alp7-Alp14 (TACC-TOG) promotes its nuclear accumulation and spindle microtubule assembly
Naoyuki Okada, Takashi Toda, Masayuki Yamamoto and Masamitsu Sato
Molecular Biology of the Cell (2014) 25(13):1969-1982. DOI:10.1091/mbc.E13-11-0679
Interdependency of fission yeast Alp14/TOG and coiled-coil protein Alp7 in microtubule localization and bipolar spindle formation
Masamitsu Sato, Leah Vardy, Miguel Angel Garcia, Nirada Koonrugsa and Takashi Toda
Molecular Biology of the Cell (2004) 15:1609-22. DOI:10.1091/mbc.e03-11-0837
Although we clarified that translocation of Alp7 affects microtubule organization as described above, it remains elusive how Alp7 promotes microtubule formation. We are currently investigating the solid mechanism as to how Alp7 contributes to the formation of microtubules.
Our research also focuses on the γ-tubulin complex, which is also known to have an important role in microtubule formation. The γ-tubulin complex is composed of γ-tubulin and its associated proteins, and is widely conserved in eukaryotes, ranging from yeast to human. It serves as a scaffold when a tubular microtubule consisting of 13 protofilaments is assembled (Fig. 3-4). Localization of the γ-tubulin complex at the spindle pole body (SPB; the centrosome equivalent in yeast) is required for spindle assembly, and we have pointed out that Pcp1, an essential factor for the localization of the γ-tubulin complex, may interact with Alp7. There are other spindle regulators such as Msd1, which interact with the γ-tubulin complex, and the relation between Alp7 and those factors is also under investigation.
Figure 3-4 The γ-tubulin complex is the base for microtubule nucleation
The γ-tubulin complex is composed of γ-tubulin and its associating proteins such as GCP2 and GCP3. These proteins provide a scaffold for building up a microtubule filament. (Left) Thirteen molecules of γ-tubulin provide a ring-shaped base for microtubule nucleation. α/β-tubulin dimers are brought to the ring of γ-tubulin, forming a launcher for microtubule assembly. (Right) Successive association of the tubulin dimers leads to polymerization of a microtubule filament.
Related Publication:
Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast
Ngang Heok Tang, Naoyuki Okada, Chii Shyang Fong, Kunio Arai, Masamitsu Sato and Takashi Toda
FEBS Letters (2014) 588(17):2814-21. DOI:10.1016/j.febslet.2014.06.027
Fission yeast Pcp1 links polo kinase-mediated mitotic entry to γ-tubulin-dependent spindle formation
Chii Shyang Fong, Masamitsu Sato and Takashi Toda
EMBO Journal (2010) 29:120-30. DOI:10.1038/emboj.2009.331
Interdependency of fission yeast Alp14/TOG and coiled-coil protein Alp7 in microtubule localization and bipolar spindle formation
Masamitsu Sato, Leah Vardy, Miguel Angel Garcia, Nirada Koonrugsa and Takashi Toda
Molecular Biology of the Cell (2004) 15:1609-22. DOI:10.1091/mbc.e03-11-0837
As the evidence for the molecular mechanisms underlying reorganization of microtubules in fission yeast, we are now pursuing whether a similar system operates in human cells. TACC3 protein, a human orthologue of Alp7 could be a key factor. The mechanisms for microtubule assembly in higher organisms is considered to be more complex than in yeast, and therefore, other related factors should also be investigated.
