[Show abstract][Hide abstract] ABSTRACT: Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells.
[Show abstract][Hide abstract] ABSTRACT: The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.
[Show abstract][Hide abstract] ABSTRACT: As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. Cytoplasmic microtubule arrays during interphase are reorganized into the spindle at mitotic onset. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7-Alp14 (TACC-TOG), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here, we demonstrate that Alp7-Alp14 is excluded from the nucleus during interphase using the nuclear export signal (NES) in Alp14, but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal (NLS) of Alp7, and the phospho-deficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus, our results revealed one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7-Alp14 complex to localize it to the nucleus.
Molecular biology of the cell 04/2014; 25(13). DOI:10.1091/mbc.E13-11-0679 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alp14 and Alp7 colocalize by fluorescence microscopy (View interaction) Alp4 and Alp7 colocalize by fluorescence microscopy (View interaction) Alp7 and Pcp1 colocalize by fluorescence microscopy (View interaction) Alp7 physically interacts with Pcp1 by anti tag coimmunoprecipitation (View interaction) Pcp1 physically interacts with Alp7 by anti bait coip (View interaction)
[Show abstract][Hide abstract] ABSTRACT: Tethering kinetochores at spindle poles facilitates their efficient capture and segregation by microtubules at mitotic onset in yeast. During meiotic prophase of fission yeast, however, kinetochores are detached from the poles, which facilitates meiotic recombination but may cause a risk of chromosome mis-segregation during meiosis. How cells circumvent this dilemma remains unclear. Here we show that an extensive microtubule array assembles from the poles at meiosis I onset and retrieves scattered kinetochores towards the poles to prevent chromosome drift. Moreover, the microtubule-associated protein complex Alp7-Alp14 (the fission yeast orthologues of mammalian TACC-TOG) is phosphorylated by Polo kinase, which promotes its meiosis-specific association to the outer kinetochore complex Nuf2-Ndc80 of scattered kinetochores, thereby assisting in capturing remote kinetochores. Although TOG was recently characterized as a microtubule polymerase, Dis1 (the other TOG orthologue in fission yeast), together with the Dam1 complex, plays a role in microtubule shortening to pull kinetochores polewards. Thus, microtubules and their binding proteins uniquely reconstitute chromosome configuration during meiosis.
[Show abstract][Hide abstract] ABSTRACT: Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled-coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during mitosis, although its underlying mechanism remains elusive. Here, we investigate the behaviour of Alp7 and its binding partner, Alp14/TOG, throughout the cell cycle. Interestingly, Alp7 enters the nucleus during interphase but is subsequently exported to the cytoplasm by the Exportin-dependent nuclear export machinery. The continuous nuclear export of Alp7 during interphase is essential for maintaining the array-like cytoplasmic microtubule structure. The mitosis-specific nuclear accumulation of Alp7 seems to be under the control of cyclin-dependent kinase (CDK). These results indicate that the spatiotemporal regulation of microtubule formation is established by the Alp7/TACC-Alp14/TOG complex through the coordinated interplay of Ran and CDK.