2013 ASHG Awards and Addresses

Each year at the annual meeting of The American Society of Human Genetics (ASHG), addresses are given in honor of The Society and a number of award winners. A summary of each of these addresses is given below. On the following pages, we have printed the Presidential Address and the addresses for the William Allan Award, the Curt Stern Award, and the Victor A. McKusick Leadership Award. Webcasts of these addresses, as well as those of many other presentations, can be found at http://www.ashg.org.     

2012 ASHG Awards and Addresses

Each year at the annual meeting of The American Society of Human Genetics (ASHG), addresses are given in honor of the Society and a number of award winners. A summary of each of these addresses is given below. On the following pages, we have printed the Presidential Address and the addresses for the William Allan and Curt Stern Awards. Webcasts of these addresses, as well as those of many other presentations, can be found at http://www.ashg.org.     

2015 ASHG Awards and Addresses

Each year at the annual meeting of The American Society of Human Genetics (ASHG), addresses are given in honor of The Society and a number of award winners. A summary of each of these is given below. On the following pages, we have printed the presidential address and the addresses for the William Allan Award, the Curt Stern Award, and the Victor A. McKusick Leadership Award. Webcasts of these addresses, as well as those of many other presentations, can be found at http://www.ashg.org.     

2010 ASHG Awards and Addresses

Each year at the annual meeting of The American Society of Human Genetics (ASHG), addresses are given in honor of The Society and a number of award winners. A summary of each of these addresses is given below. On the next pages, we have printed the Presidential Address and the addresses for the William Allan Award and the Victor A. McKusick Leadership Award. Webcasts of these addresses, as well as webcasts of many other presentations, can be found at http://www.ashg.org.     

2009 ASHG Awards and Addresses

Each year at the annual meeting of the American Society of Human Genetics (ASHG), addresses are given in honor of the society and a number of award winners. A summary of each of these addresses is given below. On the following pages, we have printed the Presidential Address and the addresses for the William Allan Award. Many of the other addresses, accompanied by pictures of the speakers, can be found at www.ashg.org.     

Awards and Addresses Summary

Each year at the annual ASHG meeting, addresses are given in honor of the society and a number of award winners. A summary of each of these addresses is given below. On the next pages, we have printed the Presidential Address and the addresses for the William Allan Award. The other addresses, accompanied by pictures of the speakers, can be found at www.ashg.org.     

2011 ASHG Awards and Addresses

Each year at the annual meeting of The American Society of Human Genetics (ASHG), addresses are given in honor of the Society and a number of award winners. A summary of each of these addresses is given below. On the following pages, we have printed the Presidential Address and the addresses for the William Allan and Curt Stern Awards. Webcasts of these addresses, as well as those of many other presentations, can be found at http://www.ashg.org.     

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.     

Online database for mosquito (Diptera,Culicidae) occurrence records in French Guiana

A database providing information on mosquito specimens (Arthropoda: Diptera: Culicidae) collected in French Guiana is presented. Field collections were initiated in 2013 under the auspices of the CEnter for the study of Biodiversity in Amazonia (CEBA: http://www.labexceba.fr/en/). This study is part of an ongoing process aiming to understand the distribution of mosquitoes, including vector species, across French Guiana. Occurrences are recorded after each collecting trip in a database managed by the laboratory Evolution et Diversité Biologique (EDB), Toulouse, France. The dataset is updated monthly and is available online. Voucher specimens and their associated DNA are stored at the laboratory Ecologie des Forêts de Guyane (Ecofog), Kourou, French Guiana. The latest version of the dataset is accessible through EDB’s Integrated Publication Toolkit at http://130.120.204.55:8080/ipt/resource.do?r=mosquitoes_of_french_guiana or through the Global Biodiversity Information Facility data portal at http://www.gbif.org/dataset/5a8aa2ad-261c-4f61-a98e-26dd752fe1c5 It can also be viewed through the Guyanensis platform at http://guyanensis.ups-tlse.fr     

toca-1 Is in a Novel Pathway That Functions in Parallel with a SUN-KASH Nuclear Envelope Bridge to Move Nuclei in Caenorhabditis elegans

Moving the nucleus to an intracellular location is critical to many fundamental cell and developmental processes, including cell migration, differentiation, fertilization, and establishment of cellular polarity. Bridges of SUN and KASH proteins span the nuclear envelope and mediate many nuclear positioning events, but other pathways function independently through poorly characterized mechanisms. To identify and characterize novel mechanisms of nuclear migration, we conducted a nonbiased forward genetic screen for mutations that enhanced the nuclear migration defect of unc-84, which encodes a SUN protein. In Caenorhabditis elegans larvae, failure of hypodermal P-cell nuclear migration results in uncoordinated and egg-laying–defective animals. The process of P-cell nuclear migration in unc-84 null animals is temperature sensitive; at 25° migration fails in unc-84 mutants, but at 15° the migration occurs normally. We hypothesized that an additional pathway functions in parallel to the unc-84 pathway to move P-cell nuclei at 15°. In support of our hypothesis, forward genetic screens isolated eight emu (enhancer of the nuclear migration defect of unc-84) mutations that disrupt nuclear migration only in a null unc-84 background. The yc20 mutant was determined to carry a mutation in the toca-1 gene. TOCA-1 functions to move P-cell nuclei in a cell-autonomous manner. TOCA-1 is conserved in humans, where it functions to nucleate and organize actin during endocytosis. Therefore, we have uncovered a player in a previously unknown, likely actin-dependent, pathway that functions to move nuclei in parallel to SUN-KASH bridges. The other emu mutations potentially represent other components of this novel pathway.     

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.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

Evaluation of coagulation activation after Rhinovirus infection in patients with asthma and healthy control subjects: an observational study

Background

Asthma exacerbations are frequently triggered by rhinovirus infections. Both asthma and respiratory tract infection can activate haemostasis. Therefore we hypothesized that experimental rhinovirus-16 infection and asthmatic airway inflammation act in synergy on the haemostatic balance.

Methods

28 patients (14 patients with mild allergic asthma and 14 healthy non-allergic controls) were infected with low-dose rhinovirus type 16. Venous plasma and bronchoalveolar lavage fluid (BAL fluid) were obtained before and 6 days after infection to evaluate markers of coagulation activation, thrombin-antithrombin complexes, von Willebrand factor, plasmin-antiplasmin complexes, plasminogen activator inhibitor type-1, endogenous thrombin potential and tissue factor-exposing microparticles by fibrin generation test, in plasma and/or BAL fluid. Data were analysed by nonparametric tests (Wilcoxon, Mann Whitney and Spearman correlation).

Results

13 patients with mild asthma (6 females, 19-29 y) and 11 healthy controls (10 females, 19-31 y) had a documented Rhinovirus-16 infection. Rhinovirus-16 challenge resulted in a shortening of the fibrin generation test in BAL fluid of asthma patients (t = -1: 706 s vs. t = 6: 498 s; p = 0.02), but not of controls (t = -1: 693 s vs. t = 6: 636 s; p = 0.65). The fold change in tissue factor-exposing microparticles in BAL fluid inversely correlated with the fold changes in eosinophil cationic protein and myeloperoxidase in BAL fluid after virus infection (r = -0.517 and -0.528 resp., both p = 0.01).Rhinovirus-16 challenge led to increased plasminogen activator inhibitor type-1 levels in plasma in patients with asthma (26.0 ng/mL vs. 11.5 ng/mL in healthy controls, p = 0.04). Rhinovirus-16 load in BAL showed a linear correlation with the fold change in endogenous thrombin potential, plasmin-antiplasmin complexes and plasminogen activator inhibitor type-1.

Conclusions

Experimental rhinovirus infection induces procoagulant changes in the airways of patients with asthma through increased activity of tissue factor-exposing microparticles. These microparticle-associated procoagulant changes are associated with both neutrophilic and eosinophilic inflammation. Systemic activation of haemostasis increases with Rhinoviral load.

Trial registration

This trial was registered at the Dutch trial registry (http://www.trialregister.nl): NTR1677.     

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs.     

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.     

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.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

Stimulation of Chromosomal Rearrangements by Ribonucleotides

We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase ε that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201Δ, rnh202Δ, and rnh203Δ), topoisomerase 1 (TOP1Δ), and/or carrying mutant alleles of DNA polymerases ε, α, and δ. We observed an ∼7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201Δ. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases α and δ, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication.