CitSci.org: A New Model for Managing,Documenting, and Sharing Citizen Science Data

Citizen science projects have the potential to advance science by increasing the volume and variety of data, as well as innovation. Yet this potential has not been fully realized, in part because citizen science data are typically not widely shared and reused. To address this and related challenges, we built CitSci.org (see www.citsci.org), a customizable platform that allows users to collect and generate diverse datasets. We hope that CitSci.org will ultimately increase discoverability and confidence in citizen science observations, encouraging scientists to use such data in their own scientific research.     

Collection Overview: Ten Years of Wonderful Open Access Science

To mark our tenth Anniversary at PLOS Biology, we are launching a special, celebratory Tenth Anniversary PLOS Biology Collection which showcases 10 specially selected PLOS Biology research articles drawn from a decade of publishing excellent science. It also features newly commissioned articles, including thought-provoking pieces on the Open Access movement (past and present), on article-level metrics, and on the history of the Public Library of Science. Each research article highlighted in the collection is also accompanied by a PLOS Biologue blog post to extend the impact of these remarkable studies to the widest possible audience.As we celebrate 10 years of PLOS Biology, 10 years of the Public Library of Science, and 10 years of strong advocacy and trail-blazing for the Open Access movement, we mustn''t forget the real star of the show – the fantastic science that we''ve published.It''s hard to cast one''s mind back 10 years and recall the scepticism with which open access publishing was initially received. A key concern at the time was that the model would be tainted with the stigma of “vanity publishing,” and that this model, in which the author pays to publish, is incompatible with integrity, editorial rigour, and scientific excellence. As also discussed in the accompanying editorial [1], the sheer quality of the science that has appeared in PLOS Biology has been vital for dispelling this myth.Our tenth anniversary provides us with a great opportunity to celebrate all of the 1800 or so research articles published in PLOS Biology since our launch in 2003. Unable to showcase each one in turn, we turned to our Editorial Board to help us pick the top 10 research articles to feature in a special Tenth Anniversary PLOS Biology Collection (www.ploscollections.org/Biology10thAnniversary). During the month of October, we will also publish a PLOS Biologue blog post (http://blogs.plos.org/biologue/) for each of these selected research articles, trying to capture and convey what it is about them that the staff editors, the editorial board, and the authors feel is special.By now, you''re probably wondering which papers we selected. The selection is detailed in Box 1, with links to each article. If you haven''t read these articles before, we urge you to read them now and to judge for yourself. As Editorial Board Member Steve O''Rahilly put it, “I think a common theme in many of the best PLOS Biology papers is that they are rich in data that is analysed very carefully and self-critically and presented without hype. However the conclusions are important for the biological community and their insights are likely to stand the test of time.”As well as publishing research articles, PLOS Biology has a thriving Magazine section that has hosted scientific and policy debates, aired polemical and provocative views, celebrated scientific lives in obituaries, reviewed interesting books, and explored unsolved mysteries. One example of how this section has triggered productive community debate is Rosie Redfield''s Perspective on how genetics should be taught to undergraduates [2]. Yet we don''t seek just to provoke debate, but also to enlighten; take a moment to read Georgina Mace''s editorial on the current issues and debates in the sustainability sciences [3]. We also try to break down barriers between fields [4] and to promote public engagement with science [5],[6].We feel strongly that our role doesn''t end with publishing the research article itself. Instead, we aim to unpackage the fascinating discoveries published in PLOS Biology by commissioning articles that explain the significance and impact of the research we publish to audiences of varying expertise. These companion articles range from Primers, which are written by experts who contextualise research articles for those in the field; to Synopses, which are written by science writers who digest an article for our wider readership of biologists; and finally, to PLOS Biologue blog posts, which distil research discoveries for a more general scientifically engaged public. We also use social media to bring these findings to the attention of a global online audience.Of course, the continued success of PLOS Biology doesn''t rest solely on the amazing research we''ve already published; it also hinges on the ground-breaking science we strive to publish in the future. Maintaining the high quality of the biology that we publish is of vital importance to us, not least because, as Editorial Board Member Robert Insall reflects, “What I like about PLOS Biology is that it avoids other journals'' fixation on fashion and the biggest names. This means the papers PLOS Biology is publishing now will last longer and mean more in a generation''s time.”

Box 1. Research Articles Featured in the Tenth Anniversary PLOS Biology Collection

Our Editorial Board Members helped us select 10 articles from the great science published during PLOS Biology''s first decade to feature in our Tenth Anniversary Collection. Please access these articles from the list below and from our Collection page. To read the PLOS Biologue blog posts that accompany them, please go to http://blogs.plos.org/biologue/ for more information.Carmena J et al. (2003) Learning to Control a BrainMachine Interface for Reaching and Grasping by Primates  Primer: Current Approaches to the Study of Movement Control  Synopsis: Retraining the Brain to Recover Movement Brennecke J et al. (2004) Principles of MicroRNA–Target Recognition  Synopsis: Seeds of Destruction: Predicting How microRNAs Choose Their Target Voight BF et al. (2005) A Map of Recent Positive Selection in the Human Genome  Synopsis: Clues to Our Past: Mining the Human Genome for Signs of Recent Selection Palmer C et al. (2007) Development of the Human Infant Intestinal Microbiota  Synopsis: Microbes Colonize a Baby''s Gut with Distinction Levy S et al. (2007) The Diploid Genome Sequence of an Individual Human  Synopsis: A New Human Genome Sequence Paves the Way for Individualized Genomics Illingworth R et al. (2008) A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci Silva J et al. (2008) Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition  Synopsis: A Shortcut to Immortality: Rapid Reprogramming with Tissue Cells Coppé J-P et al. (2008) Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Shu X et al. (2011) A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms Bonds MH et al. (2012) Disease Ecology, Biodiversity, and the Latitudinal Gradient in Income  Synopsis: Which Came First: Burden of Infectious Disease or Poverty?     

Addgene: Making Materials Sharing “Science As Usual”

Addgene (www.addgene.org) is a nonprofit organization that facilitates biomedical research and discovery by improving access to useful research materials and information. To fulfill this mission, Addgene works with hundreds of laboratories all over the world to collect high-quality published plasmids and data for the repository that can then be distributed to academic institutions and used to further research. Biological resource centers such as Addgene are an important part of the scientific infrastructure. They play a key role in helping scientists overcome logistical barriers to sharing, improving experimental reproducibility, and optimizing use of limited resources.     

Calculating Kolmogorov Complexity from the Output Frequency Distributions of Small Turing Machines

Drawing on various notions from theoretical computer science, we present a novel numerical approach, motivated by the notion of algorithmic probability, to the problem of approximating the Kolmogorov-Chaitin complexity of short strings. The method is an alternative to the traditional lossless compression algorithms, which it may complement, the two being serviceable for different string lengths. We provide a thorough analysis for all binary strings of length and for most strings of length by running all Turing machines with 5 states and 2 symbols ( with reduction techniques) using the most standard formalism of Turing machines, used in for example the Busy Beaver problem. We address the question of stability and error estimation, the sensitivity of the continued application of the method for wider coverage and better accuracy, and provide statistical evidence suggesting robustness. As with compression algorithms, this work promises to deliver a range of applications, and to provide insight into the question of complexity calculation of finite (and short) strings.Additional material can be found at the Algorithmic Nature Group website at http://www.algorithmicnature.org. An Online Algorithmic Complexity Calculator implementing this technique and making the data available to the research community is accessible at http://www.complexitycalculator.com.     

A Comprehensive Molecular Interaction Map for Rheumatoid Arthritis

Background

Computational biology contributes to a variety of areas related to life sciences and, due to the growing impact of translational medicine - the scientific approach to medicine in tight relation with basic science -, it is becoming an important player in clinical-related areas. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Rheumatoid Arthritis (RA).

Methodology

Due to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by RA. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. This global network has then been topologically analysed, as a whole and tissue-specifically, in order to translate the experimental molecular connections into topological motifs meaningful in the identification of tissue-specific markers and targets in the diagnosis, and possibly in the therapy, of RA.

Significance

We find that some nodes in the network that prove to be topologically important, in particular AKT2, IL6, MAPK1 and TP53, are also known to be associated with drugs used for the treatment of RA. Importantly, based on topological consideration, we are also able to suggest CRKL as a novel potentially relevant molecule for the diagnosis or treatment of RA. This type of finding proves the potential of in silico analyses able to produce highly refined hypotheses, based on vast experimental data, to be tested further and more efficiently. As research on RA is ongoing, the present map is in fieri, despite being -at the moment- a reflection of the state of the art. For this reason we make the network freely available in the standardised and easily exportable .xml CellDesigner format at ‘www.picb.ac.cn/ClinicalGenomicNTW/temp.html’ and ‘www.celldesigner.org’.     

Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes.     

Asymmetric Neuroblast Divisions Producing Apoptotic Cells Require the Cytohesin GRP-1 in Caenorhabditis elegans

Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1’s GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function.     

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.     

Synapse-Assembly Proteins Maintain Synaptic Vesicle Cluster Stability and Regulate Synaptic Vesicle Transport in Caenorhabditis elegans

The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were ∼3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10- and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.     

Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.     

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Autophagy Competes for a Common Phosphatidylethanolamine Pool with Major Cellular PE-Consuming Pathways in Saccharomyces cerevisiae

Autophagy is a highly regulated pathway that selectively degrades cellular constituents such as protein aggregates and excessive or damaged organelles. This transport route is characterized by engulfment of the targeted cargo by autophagosomes. The formation of these double-membrane vesicles requires the covalent conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE). However, the origin of PE and the regulation of lipid flux required for autophagy remain poorly understood. Using a genetic screen, we found that the temperature-sensitive growth and intracellular membrane organization defects of mcd4-174 and mcd4-P301L mutants are suppressed by deletion of essential autophagy genes such as ATG1 or ATG7. MCD4 encodes an ethanolamine phosphate transferase that uses PE as a precursor for an essential step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor used to link a subset of plasma membrane proteins to lipid bilayers. Similar to the deletion of CHO2, a gene encoding the enzyme converting PE to phosphatidylcholine (PC), deletion of ATG7 was able to restore lipidation and plasma membrane localization of the GPI-anchored protein Gas1 and normal organization of intracellular membranes. Conversely, overexpression of Cho2 was lethal in mcd4-174 cells grown at restrictive temperature. Quantitative lipid analysis revealed that PE levels are substantially reduced in the mcd4-174 mutant but can be restored by deletion of ATG7 or CHO2. Taken together, these data suggest that autophagy competes for a common PE pool with major cellular PE-consuming pathways such as the GPI anchor and PC synthesis, highlighting the possible interplay between these pathways and the existence of signals that may coordinate PE flux.     

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore–microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore–microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis.     

Resection Activity of the Sgs1 Helicase Alters the Affinity of DNA Ends for Homologous Recombination Proteins in Saccharomyces cerevisiae

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair.     

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1^CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1^60–84, should be extended with flanking residues, Cnn1^25–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.