WWW.DISSERTATION.XLIBX.INFO
FREE ELECTRONIC LIBRARY - Dissertations, online materials
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 | 5 |   ...   | 15 |

«A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the ...»

-- [ Page 1 ] --

Secondary receptors of the Shh pathway

By

Astrid Carolina Alfaro

A dissertation submitted in partial satisfaction of the requirements for the degree

of Doctor of Philosophy

in

Molecular and Cell Biology

in the

Graduate Division

of the

University of California, Berkeley

Committee in charge:

Professor Henk Roelink, Chair

Professor Gian Garriga

Professor David Schaffer

Professor Dirk Hockemeyer

Spring 2015

Abstract

Secondary receptors of the Shh pathway by Astrid Carolina Alfaro Doctor of Philosophy in Molecular and Cell Biology Professor Henk Roelink, Chair The Sonic Hedgehog signaling pathway is central in vertebrate development and in several human disease states. Our current understanding of the pathway outlines a straight-forward mechanism whereby the Shh ligand is perceived by the nine-pass transmembrane receptor Ptch1. In its active state Ptch1 represses Smo, a GPCR like protein, which acts as the ultimate on and off switch of the Shh pathway. When Shh binds Ptch1, Ptch1 becomes inactivated and Smo becomes activated, subsequently leading to the downstream activation of the Shh response.

Despite this framework our understanding of how Ptch1-Shh binding leads to Smo activation remains rudimentary. To understand this crucial step of the pathway one must consider that Ptch1 exists within the Shh receptosome. The Shh receptosome, consists of several membrane bound and transmembrane proteins, which bind Shh. Delineating the function of these proteins is important for understanding how receptosome-Shh interactions lead to Shh pathway activation. Boc, Cdo and Gas1 are secondary receptors of Shh that are known agonists of the pathway. Furthermore, all three proteins are suspected to interact with Ptch1 and there collective presence is necessary for activation of the Shh pathway. Ptch2, a paralogue of Ptch1, is dispensable for development, but has been documented to act as a repressor of the Shh pathway.

We sought to further characterize the function of Ptch2, Boc, Cdo, and Gas1 in the context of Ptch1. Through in vitro experiments we have determined that Shh binding to Ptch1; or Boc, Cdo and Gas1 alone is insufficient to potentiate positive Shh signaling, however, Shh binding to Ptch1 alone is sufficient to cause Smo localization to the primary cilia, an event associatedwith active Shh signaling. Additionally, we have found that Ptch2 functions as a repressor of the Shh pathway in the absence of Ptch1, further suggesting that Ptch2 and Ptch1 share overlapping functions. Moreover, the distinct possibility remains that like other proteins of the Resistance Nodulation Family, Ptch1 and Ptch2 may interact. Thus, the simple model of Ptch1 mediated Shh reception needs to be revised to include the collective activity of Ptch2, Boc, Cdo and Gas1.

1 Acknowledgements

First and foremost I would like to thank my supervisor Henk Roelink for allowing me to join his lab and for his great guidance, mentorship and patience throughout my graduate career. I would also like to thank present and former lab members of the Roelink lab for their advice and help but mostly for their friendship. A big thank you to Dr. Lina Kwong, Dr. Marina Meyerzon, Dr. Marietta Barro, Dr. Maarten Bijlsma, Catalina Casillas, Joanna Downes, Brock Roberts, Christopher Hess, Andrea Mich, and Veena Chatti.

It should be noted that the work presented in Chapters 2, 4 and 5 is in part derived from the previously co-authored published work entitled,” Ptch2 mediates the Shh response in Ptch1-/- cells” published in the journal Development on August 1st, 2014. Credit is attributable to the following co-authors: Astrid Alfaro, Brock Roberts, Lina Kwong, Maarten Bijlsma and Henk Roelink.

i Table of Contents Abstract………………………………………………………………………………….1 Acknowledgements……………………………………………………………………. i Table of Contents……………………………………………………………………….ii List of Acronyms………………………………………………………………………..iv List of Figures…………………………………………………………………………..vi List of Tables…………………………………………………………………………...vii 1 Introduction

Sonic Hedgehog: A signaling molecule

1.1.1 Morphogens in Development

1.1.2 Hedgehog proteins

The Sonic hedgehog-signaling cascade

Hedgehog Signaling in Disease: Developmental Abnormalities.................6 1.1.3 Holoprosencephaly

1.1.4 Polydactyly

Hedgehog Signaling in Disease: Cancer

1.1.5 Cancer

Sonic hedgehog ligand production and reception

1.1.6 Shh Receptors

Non-canonical Sonic hedgehog signaling

Non-canonical Shh signaling: Non-Smo mediated non-canonical Shh responses

1.1.7 Ptch1 is a direct dependence receptor

1.1.8 Ptch1 directs cell cycle progression

1.1.9 Ptch1 src kinase interactions

Non-canonical Shh signaling: Axon guidance and migration

Patched 1 and Patched 2: Structure and Function

1.1.10 Ptch1 Structure

1.1.11 Ptch1 Function: Shh binding and Smo repression





1.1.12 Ptch1 Function: proton driven efflux pump

1.1.13 Trimerization of RND proteins

1.1.14 Ptch1 sterol pumping

1.1.15 Patched 2

Aims of this project

2 Materials and Methods

Cell culture

Expression vectors

Production of ShhN supernatant from HEK293T cells

Neural induction of AB1 mESCs

Neural induction of Ptch1-/-;Ptch2-/-;Ptch1+/- and Ptch1-/- mESCs.........21 Luciferase assays

Stable A1:SMO:GFP;Smo-/- cell lines

Smo cilial localization assays

ii In-ovo Electroporations

Immunohistochemistry

Reporter Gene Assays for β-Galactosidase

RT-PCR

Lentiviral transductions

Western blotting

RNA transient transfections

Chemotaxis assay

TALENS

Genotyping

3 Involvement of Boc, Cdo, and Gas 1 in Shh signaling

Introduction

Results

Shh-N E90A does not elicit a Shh transcriptional response

Shh-N H183A does not elicit a Shh transcriptional response

Shh-N E90A and H183A function comparably to Shh-N WT in vivo............32 4 The Shh response in Ptch1-/- cells is ligand dependent

Introduction

Results

The proton-driven antiporter activity of Ptch1 can mediate the inhibition of Smo

Neuralized Ptch1-/- embryonic stem cells remain Shh- dependent for the induction of ventral cell types

Ptch1-/-;Shh-/- cells respond to exogenous Shh

Shh chemotaxis is unaffected in the absence of Ptch1, but remains dependent on Cdon and Boc

Ptch1-/-;Ptch2-/- NEBs have a higher level of Shh pathway activation than Ptch1-/- NEBs

Expression of Ptch2 antiporter mutants induces the Shh response in vivo..49 5 Discussion

Shh binding to Ptch1 or Boc, Cdo and Gas1, alone, is insufficient for Shh pathway activation in vitro

Ptch1, Boc, Cdo, and Gas 1 binding and Smo ciliary translocation.........52 Shh-N binding mutants function in vivo

Ptch2 represses Shh signaling in the absence of Ptch1

Possible interactions of Ptch1 and Ptch2

Overlapping functions of Ptch1 and Ptch2

References

iii List of Acronyms

AcrB – Acriflavine resistance B protein ADAMs – A disintigren and metalloproteases BCC – Basal Cell Carcinoma BMP – Bone morphogenetic protein Boc – Brother of Cdo cAMP – Cyclic adenosine monophosphate Cdo – Cell adhesion molecule-related/down-regulated by oncogenes CNS – Central Nervous system CTD – Carboxy Terminal Domain Dhh – Desert Hedgehog Disp1 – Dispatched 1 DMEM – Dulbecco's Modified Eagle's medium DNA – Deoxyribonucleic acid Dpp – Decapentaplegic DRAL – Down-regulated in rhabdomyosarcoma LIM domain protein EB – Embryoid Bodies ECD – Extracellular Cysteine-rich Domain EDTA – Ethylenediaminetetraacetic acid ELISA – The enzyme-linked immunosorbent assay En – Engrailed ER – Endoplasmic reticulum ERK – Extracellular signal-regulated kinases FBS – Fetal Bovine Serum FCS – Fetal Calf Serum FGF – Fibroblast Growth Factor Gas1 – Growth Arrest Specific protein 1 GFP – Green Fluorescent Protein GPCR – G-Protein Coupled Receptor GPI – Glycosylphosphatidylinositol HH – Hamburger-Hamilton Hh – Hedgehog Hhip – Hedgehog interacting protein HPE – Holoprosencephaly HRP – Horse Radish Peroxide HS – Heparan sulfate HSPGs – Heparan sulfate proteoglycans IFE – Interfollicular Epidermis IFT – Intraflagellar Transport Ihh – Indian Hedgehog Ihog –Interference Hedgehog IP3 – Inositol triphosphate Iro – Iriqouix LDA – Ligand Dependent Antagonism iv LDS – Lithium Dodecyl Sulfate LIA – Ligand Independent Antagonism LIF – Leukemia Inhibitory Factor MB – Medulloblastoma MCS – Multiclonal site MEFs – Mouse Embryonic Fibroblasts mESCs – Mouse Embryonic Stem Cells MT – Metallothionein NBCC – Nevoid Basal Cell Carcinoma NEB – Neuralized Embryoid Body NEDD – neural precursor cell expressed developmentally downregulated protein 4 NPC1 – Niemann Pick Complex 1 P14P – phosphatidylinositol 4'-monophosphate PCR – Polymerase Chain Reaction PFA – Paraformaldehyde PKA – Protein Kinase A PLC – Protein Lipase Complex C PPD – Preaxial polydactyly PPRL – The Poisonous Plant Research Laboratory Ptch1 – Patched 1 Ptch1ΔL2– Patched 1- Shh binding loop 2 mutant Ptch1ΔL2D499A – Patched 1- Shh binding loop 2 and proton antiporter mutant Ptch2 – Patched 2 Ptch2ΔL2– Patched 2- Shh binding loop 2 mutant Ptch2ΔL2D496A – Patched 2- Shh binding loop 2 and proton antiporter mutant qPCR – quantitative Polymerase Chain Reaction RNA – Riboxynucleic acid RND – Resistance Nodulation Division RVD – Repetitive Variable Di-residue SAG – Smoothened Agonist Shh – Sonic Hedgehog ShhN – Sonic Hedgehog N- terminal domain siRNA – small interfering Riboxynucleic acid Smo – Smoothened SSD – Sterol Sensing Domain SUFU – Suppressor of Fused TALEN – Transcription Activator-Like Effector Nuclease TGFb – Transforming Growth Factor Beta UV – Ultraviolet Wg – Wingless WT – Wild type ZPA – Zone of Polarizing Activity

v List of figures

Figure 1. Shh patterns the mammalian neural tube.

Figure 2. Overview of the Shh transcriptional response Figure 3.

Shh-N E90A does not elicit a transcriptional response in a Light II assay but does cause Smo localization to the primary cilium Figure 4. Shh residue E90 is important for proper Shh pathway Figure 5. Shh-N H183A is unable to elicit a Shh transcriptional response in a Light II assay or in a Smo localization assay Figure 6. Electroporation of Shh-N mutants in vivo leads to an upregulation of the Shh response.

Figure 7. Inhibition of Smo is mediated by the proton-driven antiporter activity of Ptch1 Figure 8.

Overexpression of mPtch1D499AA and ggPtch1D513A mutants.

Figure 9. Co-electroporation of Ptch1ΔL2 and Smo-M2 or Ptch1ΔL2D499AA and Smo-M2 into HH stage 10 chick embryos.

Figure 10. The Shh-binding loop2 of Ptch1 can mediate the Shh response in Ptch1-/- fibroblasts independent of the proton-driven antiporter activity.

Figure 11. Activation of the Shh response in Ptch1-/- mESCs is induced by Shh Figure 12.

Ptch1-/-;Shh-/- cells respond to Shh Figure 13. Fibroblast chemotaxis to Shh does not require Ptch1, but is sensitive to Ptch1-mediated inhibition Figure 14. Summary of the modified Boyden chamber migration assay and analysis of results Figure 15. Fibroblast chemotaxis to rShhN requires Cdon and Boc Figure 16. Ptch1-/-;Shh-/- cells respond to Shh Figure 17. Compound Ptch1, Ptch2, and Smo mutants display activation or down-regulation of the Shh response accordingly Figure 18. Expression of Ptch2 antiporter mutants causes widespread activation of the Shh response.

–  –  –

Table 1. Primers used to mutate Shh-N into binding mutants against Boc, Cdo, Gas1, and or Ptch.

Table 2. Receptor binding partners of Shh-N WT, Shh-N E90A, and Shh-N H183A.

–  –  –

1.1.1 Morphogens in Development How complex multicellular life arises from single cellular beginnings is a core question of biology. The environments to which cells in a developing embryo are exposed to are central in determining cell fate. The molecular nature and function of these extracellular signals has always been of great interest. Morphogens are a special type of extracellular signaling molecule; cells acquire distinct phenotypes in response to different concentrations of a morphogen. Morphogens instruct cells to acquire a specific fate, either directly or at a distance in a concentration dependent fashion. Usually, graded distribution of a morphogen is established away from a local source, inducing stereotypic cell differentiation. The graded activity of morphogens dictates many of the complex structures that arise during development (Rogers and Schier 2011). Well known morphogens include protein families such as the Bone Growth and promoting factors (BMPs), Wingless (Wg), Fibroblast growth factors (FGFs), Transforming Growth Fibroblast Beta (TGFβ) and Hedgehog (Hh).

–  –  –

1 wing imaginal disc. Within this structure Hh is secreted from the posterior, where it is induced by Engrailed (En), and signals in the anterior compartment at the anteriorposterior compartment boundary. High Hh signaling at the compartment boundary leads to the expression of the protein decapentaplegic (dpp). The dpp domain presents as a stripe, which physically divides the anterior from the posterior compartment (Biehs, Sturtevant, and Bier 1998). This early patterning ultimately turns the wing imaginal disc into a fly wing appendage.

In vertebrates, hedgehog proteins govern the developmental aspects of distinct organs and tissues. Shh is involved in varying signaling centers including the zone of polarizing activity (ZPA) of the early limb buds and the ventral midline of the neural tube.



Pages:   || 2 | 3 | 4 | 5 |   ...   | 15 |


Similar works:

«TOHOKU LABORATORY OF SUSTAINABLE UNIVERSITY ENVIRONMENTAL BIOLOGY Molecular identification and epidemiological characterization of cryptosporidiosis and fasciolosis in central Vietnam (ベトナム中部におけるクリプトスポリジウム症および肝蛭症の分子疫学的研究) Nguyen Thi Sam Dissertation submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Agricultural Sciences, 2013 Advisors: Prof. Dr. Yutaka Nakai Dr. Nguyen Duc Tan Laboratory of...»

«Biosensors: Introduction Overview A biosensor is a device that in some way makes use of a biological detection system. Biosensing is the process of using biosensors to gather information about living systems. The precise interpretation of the term „biosensing‟ depends on the research domain. In embodied robotics, for example, biosensing is interpreted as the various ways in which natural systems „sense‟ their environments. Understanding these processes is important to this domain as...»

«SYSTEM SZKOLENIA MŁODZIEŻY w klubie Górnik Łęczna S.A OPRACOWAŁ: TADEUSZ ŁAPA Trener Koordynator Prezentacja w ramach obowiązków uczestnika Kursu Trenerów UEFA PRO w Szkole Trenerów PZPN w roku 2006/7 Spis treści 1.Wstęp 1.1. Cel systemu 1.2. Rada Trenerów 2.Organizacja szkolenia;2.1. Schemat szkolenia, 2.2. Nabór i selekcja, 2.3. Organizacja klas sportowych, 2.4. Opieka szkoleniowa, 2.4.1Przydział trenerów do poszczególnych zespołów. 2.4.2. Udział zawodników I zespołu...»

«Case 4:04-cv-02688-JEJ Document 309 Filed 11/04/2005 Page 1 of 18 Appendix A: Excerpts from Of Pandas and People (2nd ed., 1993), the published version used by students, Quote A: “This book has a single goal: to present data from six areas of science that bear on the central question of biological origins. We don't propose to give final answers, nor to unveil The Truth. Our purpose, rather, is to help readers understand origins better, and to see why the data may be viewed in more than one...»

«1 Curriculum Vita John Korstad Feb. 22, 2015 Personal Data: Current Position: Professor of Biology (since Fall 1980) Department of Biology Oral Roberts University 7777 S. Lewis Tulsa, OK 74171 Phone Number: School: (918) 495-6942 Fax: (918) 495-6297 E-mail: jkorstad@oru.edu ORU Faculty Profile: http://webapps.oru.edu/new_php/academics/faculty_profile.php?id=9&k= Education: Undergraduate: California Lutheran University, Thousand Oaks, CA B.A. (Geology) and B.S. (Biology), 1972 Graduate:...»

«Development of functional biomaterials by self-assembled nanostructures. Tatsuyuki Yoshii 2014 Preface and Acknowledgements The studies presented in this dissertation have been carried out under the direction of Professor Itaru Hamachi at the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, from April 2005 to September 2014. The study is focused on the development of functional biomaterials by self-assembled nanostructures. My...»

«Title: Squamous cell carcinoma of the oral tongue in young non-smokers is genomically similar to tumors in older smokers.Authors: Curtis R Pickering1, Jiexin Zhang2, David M Neskey1, Mei Zhao1, Samar A Jasser1, Jiping Wang1, Alexandra Ward1, C Jillian Tsai3, Marcus V Ortega Alves1, Jane H Zhou4, Jennifer Drummond6, Adel K El-Naggar4, Richard Gibbs6,7, John N Weinstein2,5, David A Wheeler6, Jing Wang2, Mitchell J Frederick1, Jeffrey N Myers1 Affiliations: 1 Department of Head and Neck Surgery,...»

«REJOINING OF DNA DOUBLE-STRAND BREAKS AND GENOME STABILITY: FROM HOST-PATHOGEN INTERACTIONS TO BREAK-INDUCED MUTAGENESIS by Zhuobin Liang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) in the University of Michigan 2015 Doctoral Committee: Associate Professor Thomas E. Wilson, Co-Chair Associate Professor Lyle Simmons, Co-Chair Associate Professor Anuj Kumar Professor Jianming Li...»

«Gun Play • Jay Mechling Biology and the particular gun culture of the United States come together to explain the persistent and powerful attraction of American boys to both real guns and toy guns. The 1990s saw adults begin to conflate “the gun problem” with “the boy problem,” sparking attempts (largely failed) to banish toy guns from homes and schools. Following the approach of play scholar Gregory Bateson, this article offers an understanding of play with guns, maintains this moral...»

«Daniel M. Weinreich Department of Ecology and Evolutionary Biology, 401/863-3937 (v) -2749 (lab) -2166 (f) and Center for Computational Molecular Biology Daniel_Weinreich@Brown.edu Brown University, Box G-W http://research.brown.edu/myresearch/ Providence, RI 02912 Daniel_Weinreich EDUCATION 1998 Ph.D. in Population Genetics and Molecular Evolution (Richard Lewontin, advisor). Harvard University, Cambridge, Massachusetts. 1983 B.S. in Computer Science, with highest honors (John H. Holland,...»

«1 Curriculum Vita John Korstad Feb. 3, 2016 Personal Data: Current Position: Professor of Biology (since Fall 1980) Department of Biology Oral Roberts University 7777 S. Lewis Tulsa, OK 74171 Phone Number: School: (918) 495-6942 Fax: (918) 495-6297 E-mail: jkorstad@oru.edu ORU Faculty Profile: http://webapps.oru.edu/new_php/academics/faculty_profile.php?id=9&k= Education: Undergraduate: California Lutheran University, Thousand Oaks, CA B.A. (Geology) and B.S. (Biology), 1972 Graduate:...»

«SOCIAL MIRRORS AND THE BRAIN including a functional imaging study of role-play and verse PhD Thesis Charles Whitehead Department of Anthropology University College London 2003 1 Abstract This thesis attempts to bridge the conceptual gulf dividing social from biological anthropology using three main lines of argument: 1. Enculturated human cooperation originated in expanded systems of kinship and reciprocity which require obfuscation of selfish bodies. Disembodied self perceptions characterize...»





 
<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.