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THE GENOMIC STRUCTURE of the ZEBRAFISH Wnt8b GE NE
THE GENOMIC STRUCTURE OF THE ZEBRAFISH wnt8b GENE by Yvonne Marie Beckham Department of Zoology Submitted in partial fulfilment of the requirements for the degree of Master of Science Faculty of Graduate Studies The University of Western Ontario London, Ontario December 1997 O Yvonne Marie Beckham, 1997 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services seMces bibliographiques 395 Weüingtaci Street 395. Ne Wellington OttawaON K1AON4 OttawaON K1AW canada canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Lhrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, districbuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur fonnat électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Screening of a zebrafish genomic library to identify the prornoter elements of the zebrafish ivnr8b gene resulted in the isolation of two clones. p8b-3H and p8b-7A. Southem blot analysis demonstrated that clone p8b- 3H contained the 3' portion of the cDNA. p8b-7A showed only weak hybridization to the wnr8b cDNA used to initially isolate this clone. -
Regulation of Dishevelled DEP Domain Swapping by Conserved Phosphorylation Sites
Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites Gonzalo J. Beitiaa,1, Trevor J. Rutherforda,1, Stefan M. V. Freunda, Hugh R. Pelhama, Mariann Bienza, and Melissa V. Gammonsa,2 aMedical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, CB2 0QH, United Kingdom Edited by Roeland Nusse, Stanford University School of Medicine, Stanford, CA, and approved May 13, 2021 (received for review February 20, 2021) Wnt signals bind to Frizzled receptors to trigger canonical and with these effectors even if present at a low cellular concentra- noncanonical signaling responses that control cell fates during tion (1, 3, 12). animal development and tissue homeostasis. All Wnt signals are The DEP domain is a small globular domain composed of three relayed by the hub protein Dishevelled. During canonical (β-catenin– α-helices and a flexible hinge loop between the first (H1) and sec- dependent) signaling, Dishevelled assembles signalosomes via dy- ond helix (H2), which, in the monomeric configuration, folds back namic head-to-tail polymerization of its Dishevelled and Axin (DIX) on itself to form a prominent “DEP finger” that is responsible domain, which are cross-linked by its Dishevelled, Egl-10, and Pleck- for binding to Frizzled (Fig. 1A) (13). DEP dimerization involves strin (DEP) domain through a conformational switch from monomer a highly unusual mechanism called “domain swapping” (14). During to domain-swapped dimer. The domain-swapped conformation of this process, H1 of one DEP monomer is exchanged with H1 from a DEP masks the site through which Dishevelled binds to Frizzled, im- reciprocal one through outward motions of the hinge loops, replacing plying that DEP domain swapping results in the detachment of Dish- intra- with intermolecular contacts. -
AXIN1 Protein Full-Length Recombinant Human Protein Expressed in Sf9 Cells
Catalog # Aliquot Size A71-30G-20 20 µg A71-30G-50 50 µg AXIN1 Protein Full-length recombinant human protein expressed in Sf9 cells Catalog # A71-30G Lot # E3330-6 Product Description Purity Full-length recombinant human AXIN1 was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. This gene accession number is BC044648. The purity of AXIN1 protein was Gene Aliases determined to be >75% by densitometry. AXIN; MGC52315 Approx. MW 135 kDa. Formulation Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 50mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. Storage and Stability Store product at –70oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Scientific Background AXIN 1 encodes a cytoplasmic protein which contains a regulation of G-protein signaling (RGS) domain and a dishevelled and axin (DIX) domain that interacts with adenomatosis polyposis coli, catenin beta-1, glycogen synthase kinase 3 beta, protein phosphate 2, and itself. AXIN1 has both positive and negative regulatory roles in Wnt-beta-catenin signaling. AXIN1 is a core component of a 'destruction complex' that promotes AXIN1 Protein phosphorylation and polyubiquitination of cytoplasmic beta- Full-length recombinant human protein expressed in Sf9 cells catenin, resulting in beta-catenin proteasomal degradation in the absence of Wnt signaling. Nuclear accumulation of AXIN1 can Catalog # A71-30G positively influence beta-catenin-mediated transcription during Lot # E3330-6 Wnt signalling and can induce apoptosis (1). -
The Wnt Pathway Scaffold Protein Axin Promotes Signaling Specificity by Suppressing Competing Kinase Reactions
bioRxiv preprint doi: https://doi.org/10.1101/768242; this version posted September 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The Wnt pathway scaffold protein Axin promotes signaling specificity by suppressing competing kinase reactions Maire Gavagan1,2, Erin Fagnan1,2, Elizabeth B. Speltz1, and Jesse G. Zalatan1,* 1Department of Chemistry, University of Washington, Seattle, WA 98195, USA 2These authors contributed equally to this work *Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/768242; this version posted September 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract GSK3β is a multifunctional kinase that phosphorylates β-catenin in the Wnt signaling network and also acts on other protein targets in response to distinct cellular signals. To test the long-standing hypothesis that the scaffold protein Axin specifically accelerates β-catenin phosphorylation, we measured GSK3β reaction rates with multiple substrates in a minimal, biochemically-reconstituted system. We observed an unexpectedly small, ~2-fold Axin-mediated rate increase for the β-catenin reaction. The much larger effects reported previously may have arisen because Axin can rescue GSK3β from an inactive state that occurs only under highly specific conditions. -
Yuri Gagarin Is Required for Actin, Tubulin and Basal Body Functions in Drosophila Spermatogenesis
1926 Research Article yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis Michael J. Texada, Rebecca A. Simonette, Cassidy B. Johnson, William J. Deery and Kathleen M. Beckingham* Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 March 2008 Journal of Cell Science 121, 1926-1936 Published by The Company of Biologists 2008 doi:10.1242/jcs.026559 Summary Males of the genus Drosophila produce sperm of remarkable the yuri mutant, late clusters of syncytial nuclei are deformed length. Investigation of giant sperm production in Drosophila and disorganized. The basal bodies are also mispositioned on melanogaster has demonstrated that specialized actin and the nuclei, and the association of a specialized structure, the microtubule structures play key roles. The gene yuri gagarin centriolar adjunct (CA), with the basal body is lost. Some of (yuri) encodes a novel protein previously identified through its these nuclear defects might underlie a further unexpected role in gravitaxis. A male-sterile mutation of yuri has revealed abnormality: sperm nuclei occasionally locate to the wrong ends roles for Yuri in the functions of the actin and tubulin structures of the spermatid cysts. The structure of the axonemes that grow of spermatogenesis. Yuri is a component of the motile actin cones out from the basal bodies is affected in the yuri mutant, that individualize the spermatids and is essential for their suggesting a possible role for the CA in axoneme formation. formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm Key words: Drosophila, Spermatogenesis, Actin, Tubulin, Basal nuclei thought to strengthen the nuclei during elongation. -
EGFR Confers Exquisite Specificity of Wnt9a-Fzd9b Signaling in Hematopoietic Stem Cell Development
bioRxiv preprint doi: https://doi.org/10.1101/387043; this version posted August 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Grainger, et al, 2018 EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development Stephanie Grainger1, Nicole Nguyen1, Jenna Richter1,2, Jordan Setayesh1, Brianna Lonquich1, Chet Huan Oon1, Jacob M. Wozniak2,3,4, Rocio Barahona1, Caramai N. Kamei5, Jack Houston1,2, Marvic Carrillo-Terrazas3,4, Iain A. Drummond5,6, David Gonzalez3.4, Karl Willert#,¥,1, and David Traver¥,1,7. ¥co-corresponding authors: [email protected]; [email protected] #Lead contact 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, 92037, USA. 2Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California, 92037, USA. 3Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California, 92093, USA. 4Department of Pharmacology, University of California, San Diego, La Jolla, California, 92092 5Massachusetts General Hospital Nephrology Division, Charlestown, Massachusetts, 02129, USA. 6Harvard Medical School, Department of Genetics, Boston MA 02115 7Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, 92037, USA. Running title: A mechanism for Wnt-Fzd specificity in hematopoietic stem cells Keywords: hematopoietic stem cell (HSC), Wnt, Wnt9a, human, zebrafish, Fzd, Fzd9b, FZD9, EGFR, APEX2 1 bioRxiv preprint doi: https://doi.org/10.1101/387043; this version posted August 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. -
Multi-Modal Meta-Analysis of 1494 Hepatocellular Carcinoma Samples Reveals
Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-0088 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Multi-modal meta-analysis of 1494 hepatocellular carcinoma samples reveals significant impact of consensus driver genes on phenotypes Kumardeep Chaudhary1, Olivier B Poirion1, Liangqun Lu1,2, Sijia Huang1,2, Travers Ching1,2, Lana X Garmire1,2,3* 1Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI 96813, USA 2Molecular Biosciences and Bioengineering Graduate Program, University of Hawaii at Manoa, Honolulu, HI 96822, USA 3Current affiliation: Department of Computational Medicine and Bioinformatics, Building 520, 1600 Huron Parkway, Ann Arbor, MI 48109 Short Title: Impact of consensus driver genes in hepatocellular carcinoma * To whom correspondence should be addressed. Lana X. Garmire, Department of Computational Medicine and Bioinformatics Medical School, University of Michigan Building 520, 1600 Huron Parkway Ann Arbor-48109, MI, USA, Phone: +1-(734) 615-5510 Current email address: [email protected] Grant Support: This research was supported by grants K01ES025434 awarded by NIEHS through funds provided by the trans-NIH Big Data to Knowledge (BD2K) initiative (http://datascience.nih.gov/bd2k), P20 COBRE GM103457 awarded by NIH/NIGMS, NICHD R01 HD084633 and NLM R01LM012373 and Hawaii Community Foundation Medical Research Grant 14ADVC-64566 to Lana X Garmire. 1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-0088 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. -
Liquid Network Connectivity Regulates the Stability and Composition Of
Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components Jorge R. Espinosaa,b,c, Jerelle A. Josepha,b,c , Ignacio Sanchez-Burgosa,b,c , Adiran Garaizara,b,c , Daan Frenkelb , and Rosana Collepardo-Guevaraa,b,c,1 aMaxwell Centre, Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE, United Kindgdom; bDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; and cDepartment of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom Edited by Ken A. Dill, Stony Brook University, Stony Brook, NY, and approved April 17, 2020 (received for review October 8, 2019) One of the key mechanisms used by cells to control the spatiotem- 10), cellular bodies (11), or otherwise (8)—are ubiquitous within poral organization of their many components is the formation and the cell interior. Biomolecular condensates account for numer- dissolution of biomolecular condensates through liquid–liquid ous highly diverse domains, both inside the cytoplasm [e.g., P phase separation (LLPS). Using a minimal coarse-grained model granules (12) and RNA granules/bodies (13–15)] and in the cell that allows us to simulate thousands of interacting multivalent nucleus [e.g., Cajal bodies (16), nucleoli (17), heterochromatin proteins, we investigate the physical parameters dictating the domains (18, 19), the transport channels in the nuclear pore stability and composition of multicomponent biomolecular con- complex (20), and possibly superenhancers -
Recruitment of Adenomatous Polyposis Coli and B-Catenin to Axin-Puncta
Oncogene (2008) 27, 5808–5820 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Recruitment of adenomatous polyposis coli and b-catenin to axin-puncta MC Faux, JL Coates, B Catimel, S Cody, AHA Clayton, MJ Layton and AW Burgess Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Victoria, Australia The adenomatous polyposis coli (APC)tumour suppressor coli (APC) form a complex that promotes the phos- is a multifunctional protein involved in the regulation of phorylation of b-catenin by glycogen synthase kinase-3-b Wnt signalling and cytoskeletal dynamics. Little is known (GSK3-b), which consequently targets b-catenin for about how APC controls these disparate functions. In this ubiquitination by bTrCP and destruction by the study, we have used APC- and axin-fluorescent fusion proteasome (Aberle et al., 1997; Orford et al., 1997; proteins to examine the interactions between these Kitagawa et al., 1999). Wnt stimulation and APC proteins and show that the functionally distinct popula- mutations disrupt the APC/axin complex and result in tions of APC are also spatially separate. Axin-RFP forms increased levels of b-catenin in the nucleus and cytoplasmic punctate structures, similar to endogenous cytoplasm (Munemitsu et al., 1995). b-Catenin is known axin puncta. Axin-RFP recruits b-catenin destruction to interact with members of the Tcf family of transcrip- complex proteins, including APC, b-catenin, glycogen tion factors to activate transcription of genes involved in synthase kinase-3-b (GSK3-b)and casein kinase-1- a proliferation and cellular transformation (Korinek (CK1-a). -
Towards an Integrated View of Wnt Signaling in Development Renée Van Amerongen and Roel Nusse*
HYPOTHESIS 3205 Development 136, 3205-3214 (2009) doi:10.1242/dev.033910 Towards an integrated view of Wnt signaling in development Renée van Amerongen and Roel Nusse* Wnt signaling is crucial for embryonic development in all animal Notably, components at virtually every level of the Wnt signal species studied to date. The interaction between Wnt proteins transduction cascade have been shown to affect both β-catenin- and cell surface receptors can result in a variety of intracellular dependent and -independent responses, depending on the cellular responses. A key remaining question is how these specific context. As we discuss below, this holds true for the Wnt proteins responses take shape in the context of a complex, multicellular themselves, as well as for their receptors and some intracellular organism. Recent studies suggest that we have to revise some of messengers. Rather than concluding that these proteins are shared our most basic ideas about Wnt signal transduction. Rather than between pathways, we instead propose that it is the total net thinking about Wnt signaling in terms of distinct, linear, cellular balance of signals that ultimately determines the response of the signaling pathways, we propose a novel view that considers the receiving cell. In the context of an intact and developing integration of multiple, often simultaneous, inputs at the level organism, cells receive multiple, dynamic, often simultaneous and of both Wnt-receptor binding and the downstream, sometimes even conflicting inputs, all of which are integrated to intracellular response. elicit the appropriate cell behavior in response. As such, the different signaling pathways might thus be more intimately Introduction intertwined than previously envisioned. -
Cytoskeleton Cytoskeleton
CYTOSKELETON CYTOSKELETON The cytoskeleton is composed of three principal types of protein filaments: actin filaments, intermediate filaments, and microtubules, which are held together and linked to subcellular organelles and the plasma membrane by a variety of accessory proteins Muscle Contraction • Skeletal muscles are bundles of muscle fibers • Most of the cytoplasm consists of myofibrils, which are cylindrical bundles of two types of filaments: thick filaments of myosin (about 15 run in diameter) and thin filaments of actin (about 7 nm in diameter). • Each myofibril is organized as a chain of contractile units called sarcomeres, which are responsible for the striated appearance of skeletal and cardiac muscle. Structure of muscle cells Sarcomere • The ends of each sarcomere are defined by the Z disc. • Within each sarcomere, dark bands (called A bands because they are anisotropic when viewed with polarized light) alternate with light bands (called I bands for isotropic). • The I bands contain only thin (actin) filaments, whereas the A bands contain thick (myosin) filaments. • The myosin and actin filaments overlap in peripheral regions of the A band, whereas a middle region (called the H zone) contains only myosin. Muscle contraction • The basis for understanding muscle contraction is the sliding filament model, first proposed in 1954 both by Andrew Huxley and Ralph Niedergerke and by Hugh Huxley and Jean Hanson • During muscle contraction each sarcomere shortens, bringing the Z discs closer together. • There is no change in the width of the A band, but both the I bands and the H zone almost completely disappear. • These changes are explained by the actin and myosin filaments sliding past one another so that the actin filaments move into the A band and H zone. -
Deletions of AXIN1, a Component of the WNT/Wingless Pathway, in Sporadic Medulloblastomas1
[CANCER RESEARCH 61, 7039–7043, October 1, 2001] Advances in Brief Deletions of AXIN1, a Component of the WNT/wingless Pathway, in Sporadic Medulloblastomas1 R. P. Dahmen, A. Koch, D. Denkhaus, J. C. Tonn, N. So¨rensen, F. Berthold, J. Behrens, W. Birchmeier, O. D. Wiestler, and T. Pietsch2 Department of Neuropathology, University of Bonn Medical Center, D-53105 Bonn, Germany [R. P. D., A. K., D. D., O. D. W., T. P.]; Department of Neurosurgery at the Ludwig Maximilian University in Munich, D-81377 Munich, Germany [J. C. T.]; Department of Pediatric Neurosurgery, University of Wu¨rzburg, D-97080 Wu¨rzburg, Germany [N. S.]; Department of Pediatric Hematology/Oncology, University of Cologne, D-50924 Cologne, Germany [F. B.]; Department of Experimental Medicine II, University of Erlangen- Nu¨rnberg, D-91054 Erlangen, Germany [J. B.]; and Max-Delbru¨ck-Center for Molecular Medicine, D-13092 Berlin, Germany [W. B.] Abstract particular, mutations of -catenin and APC lead to constitutive sta- bilization of -catenin. Mutations of -catenin (5% of cases) and APC Medulloblastoma (MB) represents the most frequent malignant brain (4%) as components of the WNT pathway have been identified pre- tumor in children. Most MBs appear sporadically; however, their inci- viously in sporadic MBs (20–24). dence is highly elevated in two inherited tumor predisposition syndromes, Gorlin’s and Turcot’s syndrome. The genetic defects responsible for these Axin was initially identified as the gene product of the mouse fused diseases have been identified. Whereas Gorlin’s syndrome patients carry locus, which negatively regulates WNT signaling (25). Recent exper- germ-line mutations in the patched (PTCH) gene, Turcot’s syndrome iments demonstrate that Axin functions as a tumor suppressor in HCC patients with MBs carry germ-line mutations of the adenomatous polyposis (26).