Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin

Total Page:16

File Type:pdf, Size:1020Kb

Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin BASIC RESEARCH www.jasn.org Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin Jason L. Bakeberg,* Rachaneekorn Tammachote,† John R. Woollard,* Marie C. Hogan,* ʈ Han-Fang Tuan,* Ming Li,‡ Jan M. van Deursen,‡ Yanhong Wu, Bing Q. Huang,§ Vicente E. Torres,* Peter C. Harris,* and Christopher J. Ward* *Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota; †Department of Botany, Faculty of Science, Chulalongkorn University, Thailand; ‡Department of Pediatric and Adolescent Medicine and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota; §Miles and Shirley Fiterman Center for ʈ Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota ABSTRACT Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15–550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin. J Am Soc Nephrol 22: 2266–2277, 2011. doi: 10.1681/ASN.2010111173 Autosomal recessive polycystic kidney disease and small (100nm diameter) membrane bound (ARPKD MIM ID #263200) is characterized by di- particles which are shed into urine and bile, known lation of both the collecting ducts (CDs) in the kid- as PKD exosome–like vesicles (PKD–ELVs).6–10 It ney and hepatic fibrosis with or without nonobstruc- has been postulated that the mouse Pkhd1 and hu- tive biliary dilation, affecting 1:20,000 live births.1–3 The gene responsible for ARPKD is the polycystic kid- Received November 16, 2010. Accepted May 26, 2011. ney and hepatic disease gene (PKHD1: 472kb), which is encoded on chromosome 6p12.2, has 67 exons and Published online ahead of print. Publication date available at www.jasn.org. a full length mRNA of 16,235bp (mouse mRNA is 12,928bp).4 Fibrocystin/polyductin, the product of Correspondence: Dr. Christopher Ward, Division of Nephrol- ogy and Hypertension, Mayo Clinic, 703 Stabile Building, 200 the PKHD1 gene is a large type I membrane protein First Street SW, Rochester, MN 55905. Phone: 507-266-3050; of 4074 amino acids with a pro-protein convertase Fax: 507-266-9315; E-mail: ward.christopher@mayo. site between residues 3617..3620.5 Fibrocystin has edu been localized to the primary cilium, the basal body Copyright © 2011 by the American Society of Nephrology 2266 ISSN : 1046-6673/2211-2266 J Am Soc Nephrol 22: 2266–2277, 2011 www.jasn.org BASIC RESEARCH man PKHD1genes undergo extensive differential splicing and the possibility that the smaller mRNAs of 9, 7.7 and 7.5kb that many of the putative fibrocystin products are secreted as represent differentially spliced forms of Pkhd1, but RT–PCR they have a signal peptide but no membrane anchor.11–13 If on mouse kidney poly–A mRNA showed that overlapping RT– so, these splice forms could be partially functional or antago- PCR products from exons 1–21, 19–34, 32–52, 48–52 and nistic to full length fibrocystin function and so mutations af- 48–67 only produced products of the predicted size, with only fecting a subset of splice forms are also thought to be respon- very faint products of different molecular mass. As there is only sible for some of the phenotypic variation seen in human one Pkhd1 promoter,18 these data suggests that there is one ARPKD and the range of renal phenotypes in the available full–length form of Pkhd1 mRNA at 13kb and that the smaller mouse models.14–18 However, there is debate as to the extent of forms are the products of premature poly–adenylation. We PKHD1 and Pkhd1 splicing, since Pkhd1 has a relatively simple sequenced all of the RT-PCR products and showed that they band pattern on northern blotting.4 Here we re-investigate the were 100% identical to the published full length cDNA se- splicing of Pkhd1 at an mRNA level. Furthermore, to investi- quence NM_153179 GI:126157465. There was no evidence of gate splicing at a protein level we developed mouse models of heterogeneity, deletions or insertions implying that there was ARPKD where the Pkhd1 gene is initially transcriptionally si- no differential splicing. However, it remains possible that there lenced by a STOP cassette flanked by two loxP sites, an LSL are rare minor splice forms not delineated by this RT-PCR Ϫ Ϫ module.19 Upon removal of the LSL (Pkhd1LSL( )/LSL( )) with analysis. Cre recombinase the Pkhd1 gene reactivates, producing the ϩ Pkhd1Pk( ) allele, and expresses a form of fibrocystin with two Generation of A Pkhd1 Null Mouse and An Epitope ϩ ϩ SV5–Pk tags on its extreme N–terminus. This Pkhd1Pk( )/Pk( ) Tagged Pkhd1Pk(؉)/Pk(؉) Mouse mouse is phenotypically wildtype and produces epitope tagged To investigate differential splicing at a protein level we gener- PKD–ELVs in its urine and bile. The epitope tags are inserted ated a Pkhd1 gene with an epitope tag at its extreme N–termi- into exon–3, an exon which has been shown to be present in 21 nus. To produce mice that were completely null for Pkhd1 of 22 of the putative splice forms described by Boddu.13 If mRNA we inserted a loxP flanked transcriptional STOP cas- differential splicing occurs after exon–3 and these events alter sette into intron–2. The loxP STOP loxP (LSL) cassette con- the length of the protein, these forms of fibrocystin will be tains a puromycin acetyltransferase (pac) gene and four copies detectable upon western blotting of kidney or urine. of an SV–40 viral transcriptional termination sequence that PKD–ELVs are thought to be shed from multivesicular terminates all Pkhd1 transcripts in intron 2.19 The LSL cassette bodies (MVB) and to interact with primary cilia in a rapid and can be removed by the action of the Cre recombinase, leaving specific manner.10 Such interactions have been observed in the an SbfI site and a SalI flanked loxP site in intron-2 (54bp in embryologic node by Tanaka and in the maturation of male length), 369bp 5Ј to the beginning of exon–3, allowing the gene germ cells in the epididymis, where ELVs fuse with the flagel- to restart. This results in the expression of an epitope tagged lum (modified cilium) of the maturing sperm cells.20,21 How- form of fibrocystin that has two SV5–Pk tags encoded in– ever, there is no simple assay for PKD–ELV/primary cilium frame in exon–3. These tags, 26 amino acids in length, were interactions. Here we show that Pkhd1Pk(ϩ)/Pk(ϩ) mouse urine inserted into one of the most evolutionary nonconserved re- can supply tagged PKD–ELVs that interact with WT primary gions of fibrocystin and, hence, were predicted not to influence cilia in vitro and be detected by immuno–scanning electron the function of the protein (Figure 2B). Furthermore, once the microscopy (ISEM). We further show that tagging of the en- signal peptide is cleaved, the two SV5–Pk tags are predicted to dogenous Pkhd1 gene allows the monitoring of fibrocystin in be on the extreme N–terminus of the protein, an ideal position its physiologically relevant context, in particular its glycosyla- from which to detect the native protein in tissue sections and tion, proteolytic cleavage and shedding of fibrocystin on PKD– monitor proteolytic processing, glycosylation and putative ELVs. splice forms, by western blotting (Figure 2D). Exon–3 was also chosen as it ought to be present in 95% of putative splice forms.13 RESULTS (Comparison of Pkhd1LSL(؊)/LSL(؊) and Pkhd1Pk(؉)/Pk(؉ Investigation of PKHD1 Splicing Mice Pkhd1 is highly expressed in the kidney,4,11 and resolves as a Northern blotting and RT–PCR analysis indicates that major 13kb mRNA with three smaller minor species of 9, 7.7 the Pkhd1LSL(Ϫ)/LSL(Ϫ) mouse makes no fibrocystin mRNA and 7.5kb when probed with probes encompassing exons (Figures 1 and 3). The Pkhd1LSL(Ϫ)/LSL(Ϫ) mouse, on an F10 3–13, and 22–32. A probe encompassing exons 44–50 detects inbred BALB/cJ or C57BL/6J background, develops cysts and fi- the 9kb product weakly and not the 7.7 and 7.5kb species. brosis in the liver, histologically visible at 1 mo of age, and females However, a probe encompassing exons 60–67 detects only the develop PT dilation at 6 mo of age, (Figures 4 and 5). Male mice longest 13kb product (Figure 1A). The 9, 7.7 and 7.5kb species are protected from kidney cyst development in a manner appear to be polyadenylated as they are detected on northern similar to the Pkhd1del2/del2 mice described by Woollard blots of poly–A selected mRNA (Figure 1B).
Recommended publications
  • Educational Paper Ciliopathies
    Eur J Pediatr (2012) 171:1285–1300 DOI 10.1007/s00431-011-1553-z REVIEW Educational paper Ciliopathies Carsten Bergmann Received: 11 June 2011 /Accepted: 3 August 2011 /Published online: 7 September 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Cilia are antenna-like organelles found on the (NPHP) . Ivemark syndrome . Meckel syndrome (MKS) . surface of most cells. They transduce molecular signals Joubert syndrome (JBTS) . Bardet–Biedl syndrome (BBS) . and facilitate interactions between cells and their Alstrom syndrome . Short-rib polydactyly syndromes . environment. Ciliary dysfunction has been shown to Jeune syndrome (ATD) . Ellis-van Crefeld syndrome (EVC) . underlie a broad range of overlapping, clinically and Sensenbrenner syndrome . Primary ciliary dyskinesia genetically heterogeneous phenotypes, collectively (Kartagener syndrome) . von Hippel-Lindau (VHL) . termed ciliopathies. Literally, all organs can be affected. Tuberous sclerosis (TSC) . Oligogenic inheritance . Modifier. Frequent cilia-related manifestations are (poly)cystic Mutational load kidney disease, retinal degeneration, situs inversus, cardiac defects, polydactyly, other skeletal abnormalities, and defects of the central and peripheral nervous Introduction system, occurring either isolated or as part of syn- dromes. Characterization of ciliopathies and the decisive Defective cellular organelles such as mitochondria, perox- role of primary cilia in signal transduction and cell isomes, and lysosomes are well-known
    [Show full text]
  • Synergistic Genetic Interactions Between Pkhd1 and Pkd1 Result in an ARPKD-Like Phenotype in Murine Models
    BASIC RESEARCH www.jasn.org Synergistic Genetic Interactions between Pkhd1 and Pkd1 Result in an ARPKD-Like Phenotype in Murine Models Rory J. Olson,1 Katharina Hopp ,2 Harrison Wells,3 Jessica M. Smith,3 Jessica Furtado,1,4 Megan M. Constans,3 Diana L. Escobar,3 Aron M. Geurts,5 Vicente E. Torres,3 and Peter C. Harris 1,3 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ADPKD) are genetically distinct, with ADPKD usually caused by the genes PKD1 or PKD2 (encoding polycystin-1 and polycystin-2, respectively) and ARPKD caused by PKHD1 (encoding fibrocys- tin/polyductin [FPC]). Primary cilia have been considered central to PKD pathogenesis due to protein localization and common cystic phenotypes in syndromic ciliopathies, but their relevance is questioned in the simple PKDs. ARPKD’s mild phenotype in murine models versus in humans has hampered investi- gating its pathogenesis. Methods To study the interaction between Pkhd1 and Pkd1, including dosage effects on the phenotype, we generated digenic mouse and rat models and characterized and compared digenic, monogenic, and wild-type phenotypes. Results The genetic interaction was synergistic in both species, with digenic animals exhibiting pheno- types of rapidly progressive PKD and early lethality resembling classic ARPKD. Genetic interaction be- tween Pkhd1 and Pkd1 depended on dosage in the digenic murine models, with no significant enhancement of the monogenic phenotype until a threshold of reduced expression at the second locus was breached.
    [Show full text]
  • University of Oklahoma
    UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE MACRONUTRIENTS SHAPE MICROBIAL COMMUNITIES, GENE EXPRESSION AND PROTEIN EVOLUTION A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By JOSHUA THOMAS COOPER Norman, Oklahoma 2017 MACRONUTRIENTS SHAPE MICROBIAL COMMUNITIES, GENE EXPRESSION AND PROTEIN EVOLUTION A DISSERTATION APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________ Dr. Boris Wawrik, Chair ______________________________ Dr. J. Phil Gibson ______________________________ Dr. Anne K. Dunn ______________________________ Dr. John Paul Masly ______________________________ Dr. K. David Hambright ii © Copyright by JOSHUA THOMAS COOPER 2017 All Rights Reserved. iii Acknowledgments I would like to thank my two advisors Dr. Boris Wawrik and Dr. J. Phil Gibson for helping me become a better scientist and better educator. I would also like to thank my committee members Dr. Anne K. Dunn, Dr. K. David Hambright, and Dr. J.P. Masly for providing valuable inputs that lead me to carefully consider my research questions. I would also like to thank Dr. J.P. Masly for the opportunity to coauthor a book chapter on the speciation of diatoms. It is still such a privilege that you believed in me and my crazy diatom ideas to form a concise chapter in addition to learn your style of writing has been a benefit to my professional development. I’m also thankful for my first undergraduate research mentor, Dr. Miriam Steinitz-Kannan, now retired from Northern Kentucky University, who was the first to show the amazing wonders of pond scum. Who knew that studying diatoms and algae as an undergraduate would lead me all the way to a Ph.D.
    [Show full text]
  • Jimmunol.1701087.Full.Pdf
    A Novel Pkhd1 Mutation Interacts with the Nonobese Diabetic Genetic Background To Cause Autoimmune Cholangitis This information is current as Wenting Huang, Daniel B. Rainbow, Yuehong Wu, David of September 28, 2021. Adams, Pranavkumar Shivakumar, Leah Kottyan, Rebekah Karns, Bruce Aronow, Jorge Bezerra, M. Eric Gershwin, Laurence B. Peterson, Linda S. Wicker and William M. Ridgway J Immunol published online 20 November 2017 Downloaded from http://www.jimmunol.org/content/early/2017/11/23/jimmun ol.1701087 Supplementary http://www.jimmunol.org/content/suppl/2017/11/20/jimmunol.170108 http://www.jimmunol.org/ Material 7.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 27, 2017, doi:10.4049/jimmunol.1701087 The Journal of Immunology ANovelPkhd1 Mutation Interacts with the Nonobese Diabetic Genetic Background To Cause Autoimmune Cholangitis Wenting Huang,*,1 Daniel B. Rainbow,†,1 Yuehong Wu,* David Adams,* Pranavkumar Shivakumar,‡ Leah Kottyan,x Rebekah Karns,{ Bruce Aronow,{ Jorge Bezerra,‡ M.
    [Show full text]
  • Missense Mutation in Sterile Motif of Novel Protein Samcystin Is
    Missense Mutation in Sterile ␣ Motif of Novel Protein SamCystin is Associated with Polycystic Kidney Disease in (cy/؉) Rat Joanna H. Brown,* Marie-The´re`se Bihoreau,* Sigrid Hoffmann,† Bettina Kra¨nzlin,† Iulia Tychinskaya,† Nicholas Obermu¨ ller,‡ Dirk Podlich,† Suzanne N. Boehn,† Pamela J. Kaisaki,* Natalia Megel,† Patrick Danoy,§ Richard R. Copley,* John Broxholme,* ʈ Ralph Witzgall, Mark Lathrop,§ Norbert Gretz,† and Dominique Gauguier* *The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; †Medical Research Centre, Klinikum Mannheim, University of Heidelberg, Mannheim, Germany; ‡Division of Nephrology, Medical Clinic ʈ III, University of Frankfurt, Frankfurt, Germany; §Centre National de Ge´notypage, Evry, France; and Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/؉) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealedaCtoTtransition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence.
    [Show full text]
  • PKHD1 Gene PKHD1, Fibrocystin/Polyductin
    PKHD1 gene PKHD1, fibrocystin/polyductin Normal Function The PKHD1 gene provides instructions for making a protein called fibrocystin ( sometimes known as polyductin). This protein is present in fetal and adult kidney cells, and is also present at low levels in the liver and pancreas. Fibrocystin spans the cell membrane of kidney cells, so that one end of the protein remains inside the cell and the other end projects from the outer surface of the cell. Based on its structure, fibrocystin may act as a receptor, interacting with molecules outside the cell and receiving signals that help the cell respond to its environment. This protein also may be involved in connecting cells together (adhesion), keeping cells apart (repulsion), and promoting the growth and division of cells (proliferation). Fibrocystin is also found in cell structures called primary cilia. Primary cilia are tiny, fingerlike projections that line the small tubes where urine is formed (renal tubules). Researchers believe that primary cilia play an important role in maintaining the size and structure of these tubules; however, the function of fibrocystin in primary cilia remains unclear. Health Conditions Related to Genetic Changes Polycystic kidney disease More than 270 mutations in the PKHD1 gene have been identified in people with polycystic kidney disease. These mutations cause autosomal recessive polycystic kidney disease (ARPKD), which is a severe type of the disorder that is usually evident at birth or in early infancy. PKHD1 mutations include changes in single DNA building blocks (base pairs) and insertions or deletions of a small number of base pairs in the gene. These mutations disrupt the normal structure and function of the fibrocystin protein, or lead to the production of an abnormally small, nonfunctional version of the protein.
    [Show full text]
  • Nº Ref Uniprot Proteína Péptidos Identificados Por MS/MS 1 P01024
    Document downloaded from http://www.elsevier.es, day 26/09/2021. This copy is for personal use. Any transmission of this document by any media or format is strictly prohibited. Nº Ref Uniprot Proteína Péptidos identificados 1 P01024 CO3_HUMAN Complement C3 OS=Homo sapiens GN=C3 PE=1 SV=2 por 162MS/MS 2 P02751 FINC_HUMAN Fibronectin OS=Homo sapiens GN=FN1 PE=1 SV=4 131 3 P01023 A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens GN=A2M PE=1 SV=3 128 4 P0C0L4 CO4A_HUMAN Complement C4-A OS=Homo sapiens GN=C4A PE=1 SV=1 95 5 P04275 VWF_HUMAN von Willebrand factor OS=Homo sapiens GN=VWF PE=1 SV=4 81 6 P02675 FIBB_HUMAN Fibrinogen beta chain OS=Homo sapiens GN=FGB PE=1 SV=2 78 7 P01031 CO5_HUMAN Complement C5 OS=Homo sapiens GN=C5 PE=1 SV=4 66 8 P02768 ALBU_HUMAN Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 66 9 P00450 CERU_HUMAN Ceruloplasmin OS=Homo sapiens GN=CP PE=1 SV=1 64 10 P02671 FIBA_HUMAN Fibrinogen alpha chain OS=Homo sapiens GN=FGA PE=1 SV=2 58 11 P08603 CFAH_HUMAN Complement factor H OS=Homo sapiens GN=CFH PE=1 SV=4 56 12 P02787 TRFE_HUMAN Serotransferrin OS=Homo sapiens GN=TF PE=1 SV=3 54 13 P00747 PLMN_HUMAN Plasminogen OS=Homo sapiens GN=PLG PE=1 SV=2 48 14 P02679 FIBG_HUMAN Fibrinogen gamma chain OS=Homo sapiens GN=FGG PE=1 SV=3 47 15 P01871 IGHM_HUMAN Ig mu chain C region OS=Homo sapiens GN=IGHM PE=1 SV=3 41 16 P04003 C4BPA_HUMAN C4b-binding protein alpha chain OS=Homo sapiens GN=C4BPA PE=1 SV=2 37 17 Q9Y6R7 FCGBP_HUMAN IgGFc-binding protein OS=Homo sapiens GN=FCGBP PE=1 SV=3 30 18 O43866 CD5L_HUMAN CD5 antigen-like OS=Homo
    [Show full text]
  • Case Report Novel Mutations of PKHD1 and AHI1 Identified in Two Families with Cystic Renal Disease
    Int J Clin Exp Pathol 2018;11(5):2869-2874 www.ijcep.com /ISSN:1936-2625/IJCEP0073693 Case Report Novel mutations of PKHD1 and AHI1 identified in two families with cystic renal disease Ling Hou1, Yue Du1, Mingming Zhang2, Pengjun Su3, Chengguang Zhao1, Yubin Wu1 Departments of 1Pediatric Nephrology and Rheumatology, 2Pathology, 3Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang, China Received January 30, 2018; Accepted March 14, 2018; Epub May 1, 2018; Published May 15, 2018 Abstract: Objective: To report newly identified mutations in two families in China with cystic renal disease. Case presentations: Two fetuses were found by prenatal ultrasound to have symmetrically enlarged kidneys with in- creased echogenicity and cystic changes. We isolated fetal and parental genomic DNAs from umbilical cord blood and circulating leukocytes, performed next generation sequencing for mutations, followed by Sanger sequencing for confirmation. We discovered two new heterozygous mutations in PKHD1: c.2507_2515delTGAAGGAGG (p.Val836_ Glu838del) in exon 24 among the fetus and father, as well as c.6840G>A (p.Trp2280*) in exon 42 among the fetus and mother. A mutation of c.2507_2515delTGAAGGAGG caused deletion of three amino acids. Two heterozygous mutations in AHI1, c.1304G>A (p.Arg435Gln), and c.3257A>G (p.Glu1086Gly) were identified in the second fetus, while the former was also found in the mother. The mutated locus in AHI1 is highly conserved among humans, dogs, mice, and monkeys. Conclusions: We report two newly identified mutations in PKHD1 and AHI1. An accurate genetic diagnosis is crucial for genetic counseling of parents with offspring carrying cystic renal disease.
    [Show full text]
  • Membrane Proteins Take Different Trafficking Pathways to the Primary Cilium
    University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-12-14 Membrane Proteins Take Different Trafficking Pathways to the Primary Cilium William Joseph Monis University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cell Biology Commons, and the Developmental Biology Commons Repository Citation Monis WJ. (2017). Membrane Proteins Take Different Trafficking Pathways to the Primary Cilium. GSBS Dissertations and Theses. https://doi.org/10.13028/M2GX0S. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/946 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact Lisa.Palmer@umassmed.edu. MEMBRANE PROTEINS TAKE DIFFERENT TRAFFICKING PATHWAYS TO THE PRIMARY CILIUM A Dissertation Presented By William Joseph Monis Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (DECEMBER, 14, 2017) INTERDISCIPLINARY GRADUATE PROGRAM MEMBRANE PROTEINS TAKE DIFFERENT TRAFFICKING PATHWAYS TO THE PRIMARY CILIUM A Dissertation Presented By William Joseph Monis This work was undertaken in the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program The signature of the Thesis Advisor signifies validation of Dissertation content ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Gregory J. Pazour, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Julie A.
    [Show full text]
  • A Role for Intraflagellar Transport Proteins in Mitosis: a Dissertation
    University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2013-06-18 A Role for Intraflagellar rT ansport Proteins in Mitosis: A Dissertation Alison R. Bright University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cell Biology Commons, and the Cellular and Molecular Physiology Commons Repository Citation Bright AR. (2013). A Role for Intraflagellar rT ansport Proteins in Mitosis: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M2FS4M. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/682 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact Lisa.Palmer@umassmed.edu. A ROLE FOR INTRAFLAGELLAR TRANSPORT PROTEINS IN MITOSIS A Dissertation Presented By ALISON BRIGHT Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY JUNE 18TH, 2013 INTERDISCIPLINARY GRADUATE PROGRAM A ROLE FOR INTRAFLAGELLAR TRANSPORT PROTEINS IN MITOSIS Dissertation Presented By Alison Rebecca Bright The signatures of the Dissertation Committee signify completion and approval as to style and content of the Dissertation _____________________________________________
    [Show full text]
  • Ciliopathies
    T h e new england journal o f medicine Review article Mechanisms of Disease Robert S. Schwartz, M.D., Editor Ciliopathies Friedhelm Hildebrandt, M.D., Thomas Benzing, M.D., and Nicholas Katsanis, Ph.D. iverse developmental and degenerative single-gene disor- From the Howard Hughes Medical Insti- ders such as polycystic kidney disease, nephronophthisis, retinitis pigmen- tute and the Departments of Pediatrics and Human Genetics, University of Michi- tosa, the Bardet–Biedl syndrome, the Joubert syndrome, and the Meckel gan Health System, Ann Arbor (F.H.); the D Renal Division, Department of Medicine, syndrome may be categorized as ciliopathies — a recent concept that describes dis- eases characterized by dysfunction of a hairlike cellular organelle called the cilium. Center for Molecular Medicine, and Co- logne Cluster of Excellence in Cellular Most of the proteins that are altered in these single-gene disorders function at the Stress Responses in Aging-Associated Dis- level of the cilium–centrosome complex, which represents nature’s universal system eases, University of Cologne, Cologne, for cellular detection and management of external signals. Cilia are microtubule- Germany (T.B.); and the Center for Hu- man Disease Modeling and the Depart- based structures found on almost all vertebrate cells. They originate from a basal ments of Pediatrics and Cell Biology, body, a modified centrosome, which is the organelle that forms the spindle poles Duke University Medical Center, Durham, during mitosis. The important role that the cilium–centrosome complex plays in NC (N.K.). Address reprint requests to Dr. Hildebrandt at Howard Hughes Med- the normal function of most tissues appears to account for the involvement of mul- ical Institute, Departments of Pediatrics tiple organ systems in ciliopathies.
    [Show full text]
  • Ciliary Genes in Renal Cystic Diseases
    cells Review Ciliary Genes in Renal Cystic Diseases Anna Adamiok-Ostrowska * and Agnieszka Piekiełko-Witkowska * Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, 01-813 Warsaw, Poland * Correspondence: anna.adamiok@cmkp.edu.pl (A.A.-O.); apiekielko@cmkp.edu.pl (A.P.-W.); Tel.: +48-22-569-3810 (A.P.-W.) Received: 3 March 2020; Accepted: 5 April 2020; Published: 8 April 2020 Abstract: Cilia are microtubule-based organelles, protruding from the apical cell surface and anchoring to the cytoskeleton. Primary (nonmotile) cilia of the kidney act as mechanosensors of nephron cells, responding to fluid movements by triggering signal transduction. The impaired functioning of primary cilia leads to formation of cysts which in turn contribute to development of diverse renal diseases, including kidney ciliopathies and renal cancer. Here, we review current knowledge on the role of ciliary genes in kidney ciliopathies and renal cell carcinoma (RCC). Special focus is given on the impact of mutations and altered expression of ciliary genes (e.g., encoding polycystins, nephrocystins, Bardet-Biedl syndrome (BBS) proteins, ALS1, Oral-facial-digital syndrome 1 (OFD1) and others) in polycystic kidney disease and nephronophthisis, as well as rare genetic disorders, including syndromes of Joubert, Meckel-Gruber, Bardet-Biedl, Senior-Loken, Alström, Orofaciodigital syndrome type I and cranioectodermal dysplasia. We also show that RCC and classic kidney ciliopathies share commonly disturbed genes affecting cilia function, including VHL (von Hippel-Lindau tumor suppressor), PKD1 (polycystin 1, transient receptor potential channel interacting) and PKD2 (polycystin 2, transient receptor potential cation channel). Finally, we discuss the significance of ciliary genes as diagnostic and prognostic markers, as well as therapeutic targets in ciliopathies and cancer.
    [Show full text]