Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.

Classical Genetics with Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an outstanding experimental model organism that has been exploited since the early part of the twentieth century for studies in biochemistry and genetics. It has been the premiere experimental system for modern functional genomics and continues to make important contributions to many areas of biology. Here we discuss its many virtues as an organism for classical genetic research.

Vitronectin-Based, Biomimetic Encapsulating Hydrogel Scaffolds Support Adipogenesis of Adipose Stem Cells.

Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVß5 and α1ß1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects.

Connecting the microtubule attachment status of each kinetochore to cell cycle arrest through the spindle assembly checkpoint.

Kinetochores generate a signal that inhibits anaphase progression until every kinetochore makes proper attachments to spindle microtubules. This spindle assembly checkpoint (SAC) increases the fidelity of chromosome segregation. We will review the molecular mechanisms by which kinetochores generate the SAC and extinguish the signal after making proper attachments, with the goal of identifying unanswered questions and new research directions. We will emphasize recent breakthroughs in how phosphorylation changes drive the activation and inhibition of the signal. We will also emphasize the dramatic changes in kinetochore structure that occur after attaching to microtubules and how these coordinate SAC function with microtubule attachment status. Finally, we will review the emerging cross talk between the DNA damage response and the SAC.

Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate non-randomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without mother-daughter bias in a common laboratory strain grown under routine laboratory conditions.

Sister chromatids segregate at mitosis without mother-daughter bias in Saccharomyces cerevisiae.

There is evidence accumulating for nonrandom segregation of one or more chromosomes during mitosis in different cell types. We use cell synchrony and two methods to show that all chromatids of budding yeast segregate randomly and that there is no mother-daughter bias with respect to Watson and Crick-containing strands of DNA.

A redundant function for the N-terminal tail of Ndc80 in kinetochore-microtubule interaction in Saccharomyces cerevisiae.

The N-terminal tail of Ndc80 is essential for kinetochore-microtubule binding in human cells but is not required for viability in yeast. We show that the yeast Ndc80 tail is required for timely mitotic progression and accurate chromosome segregation. The tail is essential when cells are limited for Dam1, demonstrating a redundant function for the Ndc80 and Dam1 complexes in vivo.

A conserved role for COMA/CENP-H/I/N kinetochore proteins in the spindle checkpoint.

The COMA/CENP-H/I kinetochore complex regulates microtubule dynamics at kinetochores. The complex is also required to generate spindle checkpoint signals in both yeast and human cells under conditions where Aurora B activity is compromised. Our data explain why mammalian cells treated with Aurora inhibitors still have a functional spindle assembly checkpoint (SAC), since the checkpoint signals through CENP-H/I/N. The SAC effect from depleting the CENP-H/I/N complex cannot be explained by a weakened SAC signal, and the complex has no role in the SAC response to paclitaxel. We propose a model to explain the differential response of human cells to nocodazole and paclitaxel.

Ketene-Based Route to rigid Cyclobutanediol Monomers for the Replacement of BPA in High Performance Polyesters.

Recently, polyesters based on the diol monomer 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCBDO) have been shown to exhibit excellent thermal stability, mechanical properties, and optical clarity. In particular, the ability of TMCBDO to replace bisphenol A as a diol monomer in polycarbonates and polyesters has resulted in significant commercial and academic interest in these types of monomers. Herein, we report a versatile synthetic strategy based on the dimerization of ketenes derived from the thermal treatment of Meldrum's acid for the synthesis of structurally diverse cyclobutanediol (CBDO) monomers. This strategy allows a library of CBDO monomers amenable to standard polyester polymerization procedures to be prepared and the structural diversity of these CBDO monomers provides polymers with tunable physical properties, such as glass transition temperature ranging from 120 to 230 °C. The versatility and modularity of this Meldrum's acid-based approach to substituted cyclobutanediols, combined with the ease of synthesis, will be important for the further development of high-performance polyester materials that are not based on bisphenol A.

Novel interactions between actin and the proteasome revealed by complex haploinsufficiency.

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

Use of yeast chemigenomics and COXEN informatics in preclinical evaluation of anticancer agents.

Bladder cancer metastasis is virtually incurable with current platinum-based chemotherapy. We used the novel COXEN informatic approach for in silico drug discovery and identified NSC-637993 and NSC-645809 (C1311), both imidazoacridinones, as agents with high-predicted activity in human bladder cancer. Because even highly effective monotherapy is unlikely to cure most patients with metastasis and NSC-645809 is undergoing clinical trials in other tumor types, we sought to develop the basis for use of C1311 in rational combination with other agents in bladder cancer. Here, we demonstrate in 40 human bladder cancer cells that the in vitro cytotoxicity profile for C1311 correlates with that of NSC-637993 and compares favorably to that of standard of care chemotherapeutics. Using genome-wide patterns of synthetic lethality of C1311 with open reading frame knockouts in budding yeast, we determined that combining C1311 with a taxane could provide mechanistically rational combinations. To determine the preclinical relevance of these yeast findings, we evaluated C1311 singly and in doublet combination with paclitaxel in human bladder cancer in the in vivo hollow fiber assay and observed efficacy. By applying COXEN to gene expression data from 40 bladder cancer cell lines and 30 human tumors with associated clinical response data to platinum-based chemotherapy, we provide evidence that signatures of C1311 sensitivity exist within nonresponders to this regimen. Coupling COXEN and yeast chemigenomics provides rational combinations with C1311 and tumor genomic signatures that can be used to select bladder cancer patients for clinical trials with this agent.

Interpreting spatial information and regulating mitosis in response to spindle orientation.

The spindle position checkpoint (SPOC) is a regulatory mechanism that ensures accurate segregation of chromosomes in polarized cells during mitosis. In this issue of Genes & Development, Chan and Amon (pp. 1639-1649) identify a phosphoprotein phosphatase (Rts1-PP2A) as a new member of the checkpoint in budding yeast and define its role in interpreting spatial information during mitosis.

Irc15 Is a microtubule-associated protein that regulates microtubule dynamics in Saccharomyces cerevisiae.

Microtubules are polymers composed of alpha-beta tubulin heterodimers that assemble into microtubules. Microtubules are dynamic structures that have periods of both growth and shrinkage by addition and removal of subunits from the polymer. Microtubules stochastically switch between periods of growth and shrinkage, termed dynamic instability. Dynamic instability is coupled to the GTPase activity of the beta-tubulin subunit of the tubulin heterodimer. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs) that interact with microtubules to regulate dynamic instability. MAPs in budding yeast have been identified that bind microtubule ends (Bim1), that stabilize microtubule structures (Stu2), that bundle microtubules by forming cross-bridges (Ase1), and that interact with microtubules at the kinetochore (Cin8, Kar3, Kip3). IRC15 was previously identified in four different genetic screens for mutants affecting chromosome transmission or repair [11-14]. Here we present evidence that Irc15 is a microtubule-associated protein, localizing to microtubules in vivo and binding to purified microtubules in vitro. Irc15 regulates microtubule dynamics in vivo and loss of IRC15 function leads to delayed mitotic progression, resulting from failure to establish tension between sister kinetochores.

DNA damage activates the SAC in an ATM/ATR-dependent manner, independently of the kinetochore.

The DNA damage checkpoint and the spindle assembly checkpoint (SAC) are two important regulatory mechanisms that respond to different lesions. The DNA damage checkpoint detects DNA damage, initiates protein kinase cascades, and inhibits the cell cycle. The SAC relies on kinetochore-dependent assembly of protein complexes to inhibit mitosis when chromosomes are detached from the spindle. The two checkpoints are thought to function independently. Here we show that yeast cells lacking the DNA damage checkpoint arrest prior to anaphase in response to low doses of the DNA damaging agent methyl methane sulfonate (MMS). The arrest requires the SAC proteins Mad1, Mad2, Mad3, Bub1, and Bub3 and works through Cdc20 and Pds1 but unlike the normal SAC, does not require a functional kinetochore. Mec1 (ATR) and Tel1 (ATM) are also required, independently of Chk1 and Rad53, suggesting that Mec1 and Tel1 inhibit anaphase in response to DNA damage by utilizing SAC proteins. Our results demonstrate cross-talk between the two checkpoints and suggest that assembling inhibitory complexes of SAC proteins at unattached kinetochores is not obligatory for their inhibitory activity. Furthermore, our results suggest that there are novel, important targets of ATM and ATR for cell cycle regulation.

Linking kinetochore-microtubule binding to the spindle checkpoint.

The spindle checkpoint blocks cell-cycle progression until chromosomes are properly attached to the mitotic spindle. Popular models propose that checkpoint proteins associate with kinetochores to produce a "wait anaphase" signal that inhibits anaphase. Recent data suggest that a two-state switch results from using the same kinetochore proteins to bind microtubules and checkpoint proteins. At least eight protein kinases are implicated in spindle checkpoint signaling, arguing that a traditional signal transduction cascade is integral to spindle checkpoint signaling.

Differential regulation of anaphase promoting complex/cyclosome substrates by the spindle assembly checkpoint in Saccharomyces cerevisiae.

The anaphase promoting complex (APC) targets proteins for degradation to promote progression through the cell cycle. Here we show that Clb5, an APCCdc20 substrate, is degraded when the spindle checkpoint is active, while other APCCdc20 substrates are stabilized, suggesting that APCCdc20 inhibition by the spindle checkpoint is substrate specific.