Research in the Gould laboratory centers around the topic of cell cycle progression in eukaryotes. One of the most fundamental decisions a cell makes is whether to divide itself into two daughter cells. Each round of division requires a highly coordinated series of events consisting of DNA replication (or S phase) and separation of the duplicated chromosomes and cellular constituents in mitosis (or M phase). These two phases are temporally separated by two gap phases: G1, which occurs prior to S phase, and G2, which occurs prior to M phase. Initiation and completion of DNA replication and mitosis are strictly regulated to ensure that genetic information and other critical cellular components are duplicated and then divided evenly between daughter cells with each cell cycle. Failure to properly coordinate these events can lead to cell death or genomic instability.
The basic components of the cell cycle are conserved among all eukaryotes. For this reason, we concentrate on understanding cell cycle regulation in the fission yeast Schizosaccharomyces pombe.
In this organism, we can use biochemical, molecular, and cytological techniques to study the cell division process and, most importantly, we can dissect the process genetically. Additionally, the S. pombe genome has been sequenced providing invaluable research tools.
One powerful method for studying the cell cycle involves the isolation of mutants that are defective in cell cycle progression. Although many of the conserved cell cycle components are essential for viability, we can isolate conditional mutants that grow well under one set of growth conditions (i.e. low temperature) but display a mutant phenotype under another set of conditions (i.e. high temperature). Mutants that are conditionally arrested in progress through the cell cycle were isolated in yeast and other fungi beginning some 30 years ago. Studies of these cdc (cell division cycle) mutants continue to advance our knowledge of cell cycle control in all eukaryotic species.
Regulation of Mitotic Exit
In order for cells to re-enter interphase from mitosis, Cdk1p must be inactivated. This occurs through reversal of Cdk1p phosphorylation events and the timely degradation of some proteins, such as cyclin B (Cdc13p).1. Role of the Clp1p phosphatase in mitotic exit
The Cdc14 family of phosphatases specifically reverses protein-directed phosphorylation events. In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by revsing Cdk1p-dependent phosphorylations. Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis. At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p. We have investigated the ability of S. pombe Clp1p, a Cdc14p homolog, to disrupt this autoamplification loop. We have shown that Clp1p is required to destabilize and inactiviate Cdc25p at the end of mitosis.
The indicated strains were synchronized by temperature shift. Cells were then released to the permissive temperature and samples were collected at the indicated time points. Extracts were prepared from these and processed for immunoblot analysis with antibodies to Cdc25p. The levels of total protein were determined by blotting with antibodies to Cdk1p, whose abundance does not vary during the cell cycle. Note that Cdc25p is more abundant and migrates more slowly in the absence of Clp1p.
Clp1p reverses Cdk1p-dependent phosphorylation of Cdc25p. A GST-Cdc25p fusion protein was phosphorylated by recombinant Cdk1p in vitro and subsequently incubated with the indicated amounts of MBP-C286S or MBP-Clp1p. Reactions were separated by SDS-PAGE, and analyzed by Coomassie blue staining (lower panel) and autoradiography (upper panel).
Cells lacking Clp1p delay Cdk1p inactivation at the end of mitosis. The indicated strains were synchronized by temperature shift. Cells were then released to the permissive temperature and samples were collected at the indicated time points. Note that Cdk1p activity as measure towards histone H1 is prolonged and cells remain with spindles and delay cytokinesis in the absence of Clp1p.
We concluded from this work that failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle. This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation. Our model of how Clp1p and the septation initiation network function together to combat Cdk1p activation by reversing the auto-amplification loop and promoting cytokinesis is provided below.
We are continuing to study Clp1p and how it contributes to the control of mitotic exit. One aspect of our work is to investigate the role of Clp1p phosphorylation in regulating its function.
2. Characterization of the APC
The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators including cyclin B. Components of the APC have been identified through genetic screens in both fission and budding yeasts as well as through biochemical purification coupled with mass spectrometric protein identification. With these approaches, 11 subunits of the core S. cerevisiae APC were identified.
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Table 1. TAP/DALPC results
II.Apc13p-TAP purification * Number of unique tryptic peptides identified from each protein by mass spectrometry Sc = S. cerevisiae homologue |
We applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S. pombe and S. cerevisiae APCs. Proteins isolated with Lid1p-TAP and Apc13p-TAP were visualized by silver staining of a portion of the purified complex (above). The remainder of the purification was analyzed by mass spectrometry (above).
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Table 2. TAP/DALPC result of S. cerevisiae
II. Swm1p-TAP purification III. Mnd2p-TAP purification * Number of unique tryptic peptides identified from each protein by mass spectrometry |
The same strategy was then applied to S. cerevisiae. Proteins isolated with Apc4-TAP included two previously unrecognized components (Swm1p and Mnd2p) that were then targeted for reciprocal TAP and mass spectrometry (above). Our data increased the total number of identified APC subunits to 13 in both yeasts. We also obtained data that the loss of function of these components caused defects in progression through mitosis.
We are currently examining APC composition in checkpoint-arrested cells and the phosphorylation status of APC components during mitosis.
3. Coordination between mitotic exit and cytokinesis
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. The SPB is the yeast equivalent of the centrosome. We have dissected the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. On example is shown below. In it, we have established the requirement of Cdc11p for docking the Sid2p-Mob1p protein kinase at the SPB by overproducing a dominant negative form of Cdc11p under control of the thiamine-repressible nmt1 promoter.
Our combined biochemical and localization data allow us to build a comprehensive model of SIN component organization at the SPB (shown below). A current challenge in the laboratory is to identify the targets of the kinases that regulate SIN activity, Cdk1p and the Polo-like kinase.
In addition to its role in promoting cytokinesis and septation, the SIN is required for the formation and function of the equatorial microtubule organizing center (EMTOC) that forms at the end of mitosis and for astral microtubule attachment during mitosis. In order to advance our understanding of the SIN in organizing the EMTOC, we reasoned that it is important to identify all components of the S.pombe g-TuC and proteins associated with it and to establish their roles in spindle and cytoplasmic microtubule organization. To that end, we targeted known S.pombe g-TuC components for purification and identified two previously uncharacterized proteins (Gfh1p and Mbo1p) that we establish are physically and genetically associated with them. Their absence leads to defects in astral microtubule attachment and formation, respectively (see below). A current challenge is to understand in molecular detail the roles of Gfh1p and Mbo1p in MT function and their connection to the SIN.
4. Pre-mRNA splicing
We have had a long-standing interest in the pre-mRNA splicing complexes defined by the presence of S. pombe Cdc5p and Cwf8p (called Cef1p and Prp19p in S. cerevisiae). We have undertaken collaborative projects to determine the structures of large complexes that contain these proteins and to determine the structures of pieces of these proteins. One of our efforts was with Craig Vander Kooi in Walter Chazin's laboratory to determine the U-box structure within Prp19p by NMR (see below). We found that the U-box structure is similar to that of RING-finger domains found in ubiquitin ligases but that the conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. Further structure-function analysis suggest that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo. We are currently working to identify the substrates for Prp19p ubiquitin ligase function important for pre-mRNA splicing.
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