Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.

Risk of ovarian cancer and inherited variants in relapse-associated genes.

BACKGROUND: We previously identified a panel of genes associated with outcome of ovarian cancer. The purpose of the current study was to assess whether variants in these genes correlated with ovarian cancer risk. METHODS AND FINDINGS: Women with and without invasive ovarian cancer (749 cases, 1,041 controls) were genotyped at 136 single nucleotide polymorphisms (SNPs) within 13 candidate genes. Risk was estimated for each SNP and for overall variation within each gene. At the gene-level, variation within MSL1 (male-specific lethal-1 homolog) was associated with risk of serous cancer (p = 0.03); haplotypes within PRPF31 (PRP31 pre-mRNA processing factor 31 homolog) were associated with risk of invasive disease (p = 0.03). MSL1 rs7211770 was associated with decreased risk of serous disease (OR 0.81, 95% CI 0.66-0.98; p = 0.03). SNPs in MFSD7, BTN3A3, ZNF200, PTPRS, and CCND1A were inversely associated with risk (p<0.05), and there was increased risk at HEXIM1 rs1053578 (p = 0.04, OR 1.40, 95% CI 1.02-1.91). CONCLUSIONS: Tumor studies can reveal novel genes worthy of follow-up for cancer susceptibility. Here, we found that inherited markers in the gene encoding MSL1, part of a complex that modifies the histone H4, may decrease risk of invasive serous ovarian cancer.

Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform.

MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca(2+)-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca(2+) concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca(2+) regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity.

Genetic and Molecular Basis of Encapsulation and Capsule Diversity in Kingella kingae

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

Physical and Genetic Analysis of the CUP1 Tandem Array in the Yeast Saccharomyces cerevisiae

The genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations.

In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.

In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis.

The research described in Chapter 2 is published in 2014.

FtsZ Protofilament Curvature is the Opposite of Tubulin Rings

Bacterial tubulin homolog FtsZ assembles straight protofilaments (pfs) that form the scaffold of the cytokinetic Z ring. These pfs can adopt a curved conformation forming a miniring or spiral tube 24 nm in diameter. Tubulin pfs also have a curved conformation, forming 42 nm tubulin rings. We have previously provided evidence that FtsZ generates a constriction force by switching from straight pfs to the curved conformation, generating a bending force on the membrane. In the simplest model the membrane tether, which exits from the C terminus of the globular FtsZ, would have to be on the outside of the curved pf. However, it is well established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In the present study we explored the direction of curvature of FtsZ rings by fusing large protein tags to the N or C terminus of the FtsZ globular domain. FtsZ with a protein tag on the N terminus did not assemble tubes. This was expected if the N terminus is on the inside, because the protein tags are too big to fit in the interior of the tube. FtsZ with C-terminal tags assembled normal tubes, consistent with the C terminus on the outside. The FN extension was not visible in negative stain, but thin section EM gave definitive evidence that the C-terminal tag was on the outside of the tubes. This has interesting implications for the evolution of tubulin. It seems likely that tubulin began with the curvature of FtsZ, which would have resulted in pfs curving toward the interior of a disassembling MT. Evolution not only eliminated this undesirable curvature, but managed to reverse direction to produce the outward curving rings, which is useful for pulling chromosomes.