Hirohisa Masuda

Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast

The main structural components of microtubules are α‐ and β‐tubulins. A group of proteins called cofactors are crucial in the formation of assembly‐competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature‐sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild‐type cells, resulting in altered cell shape, but is rescued by co‐overexpression of β‐tubulin. Alp1 co‐localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co‐sediments with taxol (paclitaxel)‐stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of β‐tubulin, cofactor D may play a vital role in microtubule‐dependent processes as a microtubule‐associated protein.

Fission Yeast Kinesin-8 Klp5 and Klp6 Are Interdependent for Mitotic Nuclear Retention and Required for Proper Microtubule Dynamics

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.

Localization of gene products using a chromosomally tagged GFP‐fusion library in the fission yeast Schizosaccharomyces pombe

We constructed a library of chromosomally‐tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3′‐end of a particular chromosomal ORF such that the full‐length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP‐fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to ∼80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.

Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

A microtubule nucleator γ-tubulin forms a multiprotein complex (γ-tubulin complex or γ-TuC), which localizes to the microtubule organizing centers (MTOCs). We identify fission yeast Mzt1/MOZART1 as a novel conserved stoichiometric component of the γ-TuC. Mzt1 is required for cell viability, microtubule organization, and γ-TuC localization to the MTOCs, yet the core γ-TuC assembles in its absence.

Spatiotemporal regulations of Wee1 at the G2/M transition

Wee1 is highly dynamic at the SPB during the G2/M transition. Wee1 accumulates at the nuclear face of the SPB when cyclin B–Cdc2 peaks at the SPB and disappears from the SPB during spindle assembly. This dynamic behavior of Wee1 at the SPB is important for regulation of cyclin B–Cdc2 activity and proper mitotic entry and progression.

The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe.

Alp4 is an essential component of the S. pombeγ‐tubulin complex. Overproduction of the carboxy‐terminus of Alp4 induces oscillatory nuclear movement led by the spindle pole body (SPB). The movement is not dependent on cytoplasmic dynein dhc1, or kinesin‐related proteins pkl1 and klp2. Rates of SPB movement correlate with elongation rates of microtubules (MTs) extending backwards from the moving SPB (backward‐extending MTs), showing that pushing forces exerted by backward‐extending MTs move the nucleus via the SPB. These backward‐extending MTs are more stable than those of control cells and, thus, are able to push the SPB further towards the cell end, inducing nuclear oscillation with larger amplitudes than in control cells. SPB movement is biased towards the new end of the cell where levels of the CLIP170 homolog Tip1 increase, suggesting that the movement is related to MT‐mediated cell polarity control. These results demonstrate that the carboxy‐terminus of Alp4 alters MT dynamics and induces nuclear oscillation by modulating a nuclear positioning mechanism based on the balance of MT pushing forces, and suggest that regulation of γ‐tubulin complex activity is important for controlling MT dynamics and nuclear positioning.

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilising drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.

Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic gamma-tubulin complexes.

The γ‐tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule‐mediated cellular processes. Alp4 is one of the essential components of the S. pombeγ‐tubulin complex. We show here that overproduction of a carboxy‐terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of γ‐tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic γ‐tubulin complexes. Nuclear Alp4C induces a Wee1‐dependent G2 delay, reduces the levels of the γ‐tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear γ‐tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic γ‐tubulin complex activity is important for nuclear positioning and cell polarity control during interphase.

Fission yeast nucleolar protein Dnt1 regulates G2/M transition and cytokinesis by downregulating Wee1 kinase

Summary Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive. By contrast, it is clear that its budding yeast relative, Net1/Cfi1, regulates the homologous MEN signaling pathway by sequestering Cdc14 phosphatase in the nucleolus before mitotic exit. In this study, we show that dnt1+ positively regulates G2/M transition during the cell cycle. By conducting epistasis analyses to measure cell length at septation in double mutant (for dnt1 and genes involved in G2/M control) cells, we found a link between dnt1+ and wee1+. Furthermore, we showed that elevated protein levels of the mitotic inhibitor Wee1 kinase and the corresponding attenuation in Cdk1 activity is responsible for the rescuing effect of dnt1&Dgr; on SIN mutants. Finally, our data also suggest that Dnt1 modulates Wee1 activity in parallel with SCF-mediated Wee1 degradation. Therefore, this study reveals an unexpected missing link between the nucleolar protein Dnt1 and the SIN signaling pathway, which is mediated by the Cdk1 regulator Wee1 kinase. Our findings also define a novel mode of regulation of Wee1 and Cdk1, which is important for integration of the signals controlling the SIN pathway in fission yeast.