Schizosaccharomyces pombe homolog of Survivin, Bir1p, exhibits a novel dynamic behavior at the spindle mid-zone.

Members of the BIR-domain containing Survivin family of proteins have been identified in a variety of eukaryotes and are known to play important roles in the regulation of mitosis. The Schizosaccharomyces pombe homolog of Survivin, Bir1p, is essential for chromosome condensation and spindle elongation and integrity. Bir1p, a nuclear protein, resides at the kinetochores in metaphase and anaphase A and spreads to the spindle mid-zone in anaphase B. Here we show that this relocation requires Cdk (Cyclin dependent kinase) inactivation and intact microtubules. With the aid of a kinesin mutant, klp5Delta, we also show that completion of anaphase A is vital for effecting Bir1p re-location to the spindle mid-zone. Although minimal exchange of Bir1p sub-units occurs between the spindle and the nucleoplasm, the protein redistributes laterally within the mid-zone region. Bir1p dynamics therefore significantly differs from that of tubulin on an anaphase B spindle, which is loaded at the plus ends of growing microtubules and shows no lateral redistribution within the spindle. Thus, Bir1p, and possibly its associated proteins, might organize a dynamic mid-zone region that helps spindle elongation and maintenance.

The nuclear kinase Lsk1p positively regulates the septation initiation network and promotes the successful completion of cytokinesis in response to perturbation of the actomyosin ring in Schizosaccharomyces pombe.

Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN). In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression. These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability. To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase. Similar to clp1Delta mutants, and in contrast to wild type, lsk1Delta cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 microM) of latrunculin A. However, unlike clp1Delta mutants, lsk1Delta cells are competent to delay nuclear cycle progression after cytokinetic failure. In addition, lsk1Delta mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module. In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation. Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways.

The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase.

The S. pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint. At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring. This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S. cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition. Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation. Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores. Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores.

The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe.

Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable.

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB.

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.

A potential tension-sensing mechanism that ensures timely anaphase onset upon metaphase spindle orientation.

The spindle orientation checkpoint (SOC) in fission yeast has been proposed to delay metaphase-to-anaphase transition when the spindle poles are misaligned with respect to the long axis of the cell. This checkpoint is activated in the absence of either an actomyosin division ring or astral microtubules. Although the SOC could be overridden in the absence of the transcription factor Atf1p, its mechanistic nature remained unclear. Here, we show that the SOC-triggered metaphase delay depends on a subset of the spindle assembly checkpoint (SAC) components Mph1p and Bub1p. Based on this finding and a detailed imaging of the spindle orientation process, we hypothesized that the spindle pole might contain proteins capable of sensing the achievement of spindle alignment. We identified the kendrin-like spindle pole body resident Pcp1p as a candidate molecule. A targeted mutation in its central domain specifically triggered the SOC in spite of the presence of oriented spindles, causing a metaphase delay that could be relieved in the absence of Mph1p, Bub1p, and Atf1p. Thus, Pcp1p might provide a link between the mechanical process of spindle alignment and the signal transduction that initiates anaphase.

Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe.

Sterol-rich membrane domains exist in unicellular and multicellular eukaryotes. They are thought to provide a structural framework for interactions among a subset of proteins by selectively incorporating some proteins while excluding others. Although most studies have focused on the biophysical and biochemical properties of sterol-rich membrane domains and incorporated proteins, relatively little is known about their intracellular distribution. Using a cytological approach we show here that in the fission yeast Schizosaccharomyces pombe, sterols are enriched in the plasma membrane at the growing cell tips and at the site of cytokinesis. The distribution of sterols is regulated in a cell-cycle-dependent manner and requires a functional secretory pathway. By manipulating the integrity of sterol-rich membrane domains using sterol sequestering agents and genetic means, we show that these domains are important for multiple processes regulating cytokinesis. In these cells, defects in proper maintenance of the actomyosin ring and/or its attachment to the overlying plasma membrane were observed. Furthermore, the stability of a plasma membrane protein that colocalises with sterol-rich membrane domains was compromised. Taken together, our studies establish S. pombe as a genetically tractable model organism in which to study the role(s) of sterol-rich membrane domains in cell polarity and cytokinesis.

Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis.

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.